Use of Galerina marginata genes and proteins for peptide production

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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

DOEpatents

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

2016-03-01

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.

Identification of the neurofibromatosis type 1 gene product

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gutmann, D.H.; Wood, D.L.; Collins, F.S.

The gene for neurofibromatosis type 1 (NF1) was recently identified by positional cloning. The complete cDNA encodes a polypeptide of 2818 amino acids. To study the NF1 gene product, antibodies were raised against both fusion proteins and synthetic peptides. Initial characterization of two anti-peptide antibodies and one fusion-protein antibody demonstrated a specific protein of {approx}250 kDa by both immunoprecipitation and immunoblotting. This protein was found in all tissues and cell lines examined and is detected in human, rat, and mouse tissues. To demonstrate that these antibodies specifically recognize the NF1 protein, additional fusion proteins containing the sequence specific to themore » synthetic peptide were generated. Both peptide antisera recognize the proper specific fusion proteins so generated. Immunoprecipitates using the peptide antisera were shown to recognize the same protein detected by immunoblotting with either the other peptide antiserum or the fusion-protein antiserum. Immunoblotting using antiserum specific to spatially distinct epitopes conducted on tissue homogenates demonstrated the NF1 protein in all adult tissues. Based on the homology between the NF1 gene product and members of the GTPase-activating protein (GAP) superfamily, the name NF1-GAP-related protein (NF1GRP) is suggested.« less

Additive manufacturing in production: challenges and opportunities

NASA Astrophysics Data System (ADS)

Ahuja, Bhrigu; Karg, Michael; Schmidt, Michael

2015-03-01

Additive manufacturing, characterized by its inherent layer by layer fabrication methodology has been coined by many as the latest revolution in the manufacturing industry. Due to its diversification of Materials, processes, system technology and applications, Additive Manufacturing has been synonymized with terminology such as Rapid prototyping, 3D printing, free-form fabrication, Additive Layer Manufacturing, etc. A huge media and public interest in the technology has led to an innovative attempt of exploring the technology for applications beyond the scope of the traditional engineering industry. Nevertheless, it is believed that a critical factor for the long-term success of Additive Manufacturing would be its ability to fulfill the requirements defined by the traditional manufacturing industry. A parallel development in market trends and product requirements has also lead to a wider scope of opportunities for Additive Manufacturing. The presented paper discusses some of the key challenges which are critical to ensure that Additive Manufacturing is truly accepted as a mainstream production technology in the industry. These challenges would highlight on various aspects of production such as product requirements, process management, data management, intellectual property, work flow management, quality assurance, resource planning, etc. In Addition, changing market trends such as product life cycle, mass customization, sustainability, environmental impact and localized production will form the foundation for the follow up discussion on the current limitations and the corresponding research opportunities. A discussion on ongoing research to address these challenges would include topics like process monitoring, design complexity, process standardization, multi-material and hybrid fabrication, new material development, etc.

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

PubMed

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 < 0.05). Our study shows evidence of significant associations between SNPs within the 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.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Malpass, Gloria E., E-mail: gloria.malpass@gmail.com; Arimilli, Subhashini, E-mail: sarimill@wakehealth.edu; Prasad, G.L., E-mail: prasadg@rjrt.com

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 5more » 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.« less

Dissection of additive, dominance, and imprinting effects for production and reproduction traits in Holstein cattle.

PubMed

Jiang, Jicai; Shen, Botong; O'Connell, Jeffrey R; VanRaden, Paul M; Cole, John B; Ma, Li

2017-05-30

Although genome-wide association and genomic selection studies have primarily focused on additive effects, dominance and imprinting effects play an important role in mammalian biology and development. The degree to which these non-additive genetic effects contribute to phenotypic variation and whether QTL acting in a non-additive manner can be detected in genetic association studies remain controversial. To empirically answer these questions, we analyzed a large cattle dataset that consisted of 42,701 genotyped Holstein cows with genotyped parents and phenotypic records for eight production and reproduction traits. SNP genotypes were phased in pedigree to determine the parent-of-origin of alleles, and a three-component GREML was applied to obtain variance decomposition for additive, dominance, and imprinting effects. The results showed a significant non-zero contribution from dominance to production traits but not to reproduction traits. Imprinting effects significantly contributed to both production and reproduction traits. Interestingly, imprinting effects contributed more to reproduction traits than to production traits. Using GWAS and imputation-based fine-mapping analyses, we identified and validated a dominance association signal with milk yield near RUNX2, a candidate gene that has been associated with milk production in mice. When adding non-additive effects into the prediction models, however, we observed little or no increase in prediction accuracy for the eight traits analyzed. Collectively, our results suggested that non-additive effects contributed a non-negligible amount (more for reproduction traits) to the total genetic variance of complex traits in cattle, and detection of QTLs with non-additive effect is possible in GWAS using a large dataset.

Human AZU-1 gene, variants thereof and expressed gene products

DOEpatents

Chen, Huei-Mei; Bissell, Mina

2004-06-22

A human AZU-1 gene, mutants, variants and fragments thereof. Protein products encoded by the AZU-1 gene and homologs encoded by the variants of AZU-1 gene acting as tumor suppressors or markers of malignancy progression and tumorigenicity reversion. Identification, isolation and characterization of AZU-1 and AZU-2 genes localized to a tumor suppressive locus at chromosome 10q26, highly expressed in nonmalignant and premalignant cells derived from a human breast tumor progression model. A recombinant full length protein sequences encoded by the AZU-1 gene and nucleotide sequences of AZU-1 and AZU-2 genes and variant and fragments thereof. Monoclonal or polyclonal antibodies specific to AZU-1, AZU-2 encoded protein and to AZU-1, or AZU-2 encoded protein homologs.

Epigenetic Manipulation of a Filamentous Fungus by the Proteasome-Inhibitor Bortezomib Induces the Production of an Additional Secondary Metabolite.

PubMed

VanderMolen, Karen M; Darveaux, Blaise A; Chen, Wei-Lun; Swanson, Steven M; Pearce, Cedric J; Oberlies, Nicholas H

2014-01-01

The use of epigenetic modifiers, such as histone deacetylase inhibitors and DNA methyltransferase inhibitors, has been explored increasingly as a technique to induce the production of additional microbial secondary metabolites. The application of such molecules to microbial cultures has been shown to upregulate otherwise suppressed genes, and in several cases has led to the production of new molecular structures. In this study, the proteasome inhibitor bortezomib was used to induce the production of an additional metabolite from a filamentous fungus (Pleosporales). The induced metabolite was previously isolated from a plant, but the configuration was not assigned until now; in addition, an analogue was isolated from a degraded sample, yielding a new compound. Proteasome inhibitors have not previously been used in this application and offer an additional tool for microbial genome mining.

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

PubMed

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.

Analysis of Genes for Succinoyl Trehalose Lipid Production and Increasing Production in Rhodococcus sp. Strain SD-74

PubMed Central

Inaba, Tomohiro; Tokumoto, Yuta; Miyazaki, Yusuke; Inoue, Naoyuki; Maseda, Hideaki; Nakajima-Kambe, Toshiaki; Uchiyama, Hiroo

2013-01-01

Succinoyl trehalose lipids (STLs) are promising glycolipid biosurfactants produced from n-alkanes that are secreted by Rhodococcus species bacteria. These compounds not only exhibit unique interfacial properties but also demonstrate versatile biochemical actions. In this study, three novel types of genes involved in the biosynthesis of STLs, including a putative acyl coenzyme A (acyl-CoA) transferase (tlsA), fructose-bisphosphate aldolase (fda), and alkane monooxygenase (alkB), were identified. The predicted functions of these genes indicate that alkane metabolism, sugar synthesis, and the addition of acyl groups are important for the biosynthesis of STLs. Based on these results, we propose a biosynthesis pathway for STLs from alkanes in Rhodococcus sp. strain SD-74. By overexpressing tlsA, we achieved a 2-fold increase in the production of STLs. This study advances our understanding of bacterial glycolipid production in Rhodococcus species. PMID:24038682

Functions of the gene products of Escherichia coli.

PubMed Central

Riley, M

1993-01-01

A list of currently identified gene products of Escherichia coli is given, together with a bibliography that provides pointers to the literature on each gene product. A scheme to categorize cellular functions is used to classify the gene products of E. coli so far identified. A count shows that the numbers of genes concerned with small-molecule metabolism are on the same order as the numbers concerned with macromolecule biosynthesis and degradation. One large category is the category of tRNAs and their synthetases. Another is the category of transport elements. The categories of cell structure and cellular processes other than metabolism are smaller. Other subjects discussed are the occurrence in the E. coli genome of redundant pairs and groups of genes of identical or closely similar function, as well as variation in the degree of density of genetic information in different parts of the genome. PMID:7508076

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

PubMed

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.

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

NASA Astrophysics Data System (ADS)

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.

Robust Tests for Additive Gene-Environment Interaction in Case-Control Studies Using Gene-Environment Independence.

PubMed

Liu, Gang; Mukherjee, Bhramar; Lee, Seunggeun; Lee, Alice W; Wu, Anna H; Bandera, Elisa V; Jensen, Allan; Rossing, Mary Anne; Moysich, Kirsten B; Chang-Claude, Jenny; Doherty, Jennifer A; Gentry-Maharaj, Aleksandra; Kiemeney, Lambertus; Gayther, Simon A; Modugno, Francesmary; Massuger, Leon; Goode, Ellen L; Fridley, Brooke L; Terry, Kathryn L; Cramer, Daniel W; Ramus, Susan J; Anton-Culver, Hoda; Ziogas, Argyrios; Tyrer, Jonathan P; Schildkraut, Joellen M; Kjaer, Susanne K; Webb, Penelope M; Ness, Roberta B; Menon, Usha; Berchuck, Andrew; Pharoah, Paul D; Risch, Harvey; Pearce, Celeste Leigh

2018-02-01

There have been recent proposals advocating the use of additive gene-environment interaction instead of the widely used multiplicative scale, as a more relevant public health measure. Using gene-environment independence enhances statistical power for testing multiplicative interaction in case-control studies. However, under departure from this assumption, substantial bias in the estimates and inflated type I error in the corresponding tests can occur. In this paper, we extend the empirical Bayes (EB) approach previously developed for multiplicative interaction, which trades off between bias and efficiency in a data-adaptive way, to the additive scale. An EB estimator of the relative excess risk due to interaction is derived, and the corresponding Wald test is proposed with a general regression setting under a retrospective likelihood framework. We study the impact of gene-environment association on the resultant test with case-control data. Our simulation studies suggest that the EB approach uses the gene-environment independence assumption in a data-adaptive way and provides a gain in power compared with the standard logistic regression analysis and better control of type I error when compared with the analysis assuming gene-environment independence. We illustrate the methods with data from the Ovarian Cancer Association Consortium. © The Author(s) 2017. Published by Oxford University Press on behalf of the Johns Hopkins Bloomberg School of Public Health. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Methods of cracking a crude product to produce additional crude products

DOEpatents

Mo, Weijian [Sugar Land, TX; Roes, Augustinus Wilhelmus Maria [Houston, TX; Nair, Vijay [Katy, TX

2009-09-08

A method for producing a crude product is disclosed. Formation fluid is produced from a subsurface in situ heat treatment process. The formation fluid is separated to produce a liquid stream and a first gas stream. The first gas stream includes olefins. The liquid stream is fractionated to produce one or more crude products. At least one of the crude products has a boiling range distribution from 38.degree. C. and 343.degree. C. as determined by ASTM Method D5307. The crude product having the boiling range distribution from 38.degree. C. and 343.degree. C. is catalytically cracked to produce one or more additional crude products. At least one of the additional crude products is a second gas stream. The second gas stream has a boiling point of at most 38.degree. C. at 0.101 MPa.

Characterization of the Expression of Basigin Gene Products Within the Pineal Gland of Mice.

PubMed

Tokar, Derek; van Ekeris, Leslie; Linser, Paul J; Ochrietor, Judith D

2017-08-01

The expression of Basigin gene products and monocarboxylate transporter-1 (MCT1) has been investigated within the mammalian neural retina and suggests a role for these proteins in cellular metabolism within that tissue. The purpose of the present study was to investigate the expression of these same proteins in the pineal gland of the mouse brain. Mouse pineal gland and neural retina RNA and protein were subjected to quantitative reverse transcription-polymerase chain reaction and immunoblotting analyses. In addition, paraffin-embedded sections of each tissue were analyzed for expression of Basigin gene products and MCT1 via immunohistochemistry. The results indicate that MCT1 and Basigin variant-2, but not Basigin variant-1, are expressed within the mouse pineal gland. The expression of Basigin variant-2 and MCT1 was localized to the capsule surrounding the gland. The position and relative amounts of the gene products suggest that they play a much less prominent role within the pineal gland than in the neural retina.

Regulatory Oversight of Cell and Gene Therapy Products in Canada.

PubMed

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.

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

PubMed

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

2016-09-13

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

Modern plant metabolomics: Advanced natural product gene discoveries, improved technologies, and future prospects

DOE PAGES

Sumner, Lloyd W.; Lei, Zhentian; Nikolau, Basil J.; ...

2014-10-24

Plant metabolomics has matured and modern plant metabolomics has accelerated gene discoveries and the elucidation of a variety of plant natural product biosynthetic pathways. This study highlights specific examples of the discovery and characterization of novel genes and enzymes associated with the biosynthesis of natural products such as flavonoids, glucosinolates, terpenoids, and alkaloids. Additional examples of the integration of metabolomics with genome-based functional characterizations of plant natural products that are important to modern pharmaceutical technology are also reviewed. This article also provides a substantial review of recent technical advances in mass spectrometry imaging, nuclear magnetic resonance imaging, integrated LC-MS-SPE-NMR formore » metabolite identifications, and x-ray crystallography of microgram quantities for structural determinations. The review closes with a discussion on the future prospects of metabolomics related to crop species and herbal medicine.« less

Norrie gene product is necessary for regression of hyaloid vessels.

PubMed

Ohlmann, Anne V; Adamek, Edith; Ohlmann, Andreas; Lütjen-Drecoll, Elke

2004-07-01

To investigate the nature and origin of the vitreous membranes in mice with knock-out of the Norrie gene product (ND mice). Eighty-two eyes of ND mice of different age groups (postnatal day [P]0-13 months) and 95 age-matched wild-type control mice were investigated. In vitreoretinal wholemounts and in sagittal sections, vessels and free cells were visualized by labeling for lectin. In addition, staining with a marker for macrophages (F4/80) and collagen XVIII/endostatin known to be involved in regression of hyaloid vessels was performed for light and electron microscopic investigations. Endostatin expression was confirmed by Western blot analysis. Wild-type controls showed the typical pattern of hyaloid vessels, their regression and concomitantly retinal vasculogenesis and angiogenesis. Hyaloid vessels all stained for endostatin, whereas retinal vessels remained unstained. In ND mice, 1 to 5 days after birth, the hyaloid and retinal vasculatures were comparable to that in control mice. The hyaloid vessels also stained for endostatin. Numerous F4/80-positive cells were present adjacent to the vessels. With increasing age, only a few connecting branches of the hyaloid vessels regressed. Even in old mice most of the hyaloid vessels persisted. The vessels still stained for endostatin. Retinal angiogenesis was impaired. Retrolental membranes in ND mice consist of persistent hyaloid vessels, indicating that the ND gene product is important for the process of regression of these vessels. The ND gene product neither influences endostatin expression nor the presence of macrophages.

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

PubMed

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.

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

PubMed Central

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

2017-01-01

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 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. PMID:28966922

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

PubMed Central

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

Antagonistic control of a dual-input mammalian gene switch by food additives.

PubMed

Xie, Mingqi; Ye, Haifeng; Hamri, Ghislaine Charpin-El; Fussenegger, Martin

2014-08-01

Synthetic biology has significantly advanced the design of mammalian trigger-inducible transgene-control devices that are able to programme complex cellular behaviour. Fruit-based benzoate derivatives licensed as food additives, such as flavours (e.g. vanillate) and preservatives (e.g. benzoate), are a particularly attractive class of trigger compounds for orthogonal mammalian transgene control devices because of their innocuousness, physiological compatibility and simple oral administration. Capitalizing on the genetic componentry of the soil bacterium Comamonas testosteroni, which has evolved to catabolize a variety of aromatic compounds, we have designed different mammalian gene expression systems that could be induced and repressed by the food additives benzoate and vanillate. When implanting designer cells engineered for gene switch-driven expression of the human placental secreted alkaline phosphatase (SEAP) into mice, blood SEAP levels of treated animals directly correlated with a benzoate-enriched drinking programme. Additionally, the benzoate-/vanillate-responsive device was compatible with other transgene control systems and could be assembled into higher-order control networks providing expression dynamics reminiscent of a lap-timing stopwatch. Designer gene switches using licensed food additives as trigger compounds to achieve antagonistic dual-input expression profiles and provide novel control topologies and regulation dynamics may advance future gene- and cell-based therapies. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Oxaloacetate and malate production in engineered Escherichia coli by expression of codon-optimized phosphoenolpyruvate carboxylase2 gene from Dunaliella salina.

PubMed

Park, Soohyun; Chang, Kwang Suk; Jin, Eonseon; Pack, Seung Pil; Lee, Jinwon

2013-01-01

A new phosphoenolpyruvate carboxylase (PEPC) gene of Dunaliella salina is identified using homology analysis was conducted using PEPC gene of Chlamydomonas reinhardtii and Arabidopsis thaliana. Recombinant E. coli SGJS115 with increased production of malate and oxaloacetate was developed by introducing codon-optimized phosphoenolpyruvate carboxylase2 (OPDSPEPC2) gene of Dunaliella salina. E. coli SGJS115 yielded a 9.9 % increase in malate production. In addition, E. coli SGJS115 exhibited two times increase in the yield of oxaloacetate over the E. coli SGJS114 having identified PEPC2 gene obtained from Dunaliella salina.

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

PubMed

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.

Methods for detecting additional genes underlying Alzheimer disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Locke, P.A.; Haines, J.L.; Ter-Minassian, M.

1994-09-01

Alzheimer`s disease (AD) is a complex inherited disorder with proven genetic heterogeneity. To date, genes on chromosome 21 (APP) and 14 (not yet identified) are associated with early-onset familial AD, while the APOE gene on chromosome 19 is associated with both late onset familial and sporadic AD and early onset sporadic AD. Although these genes likely account for the majority of AD, many familial cases cannot be traced to any of these genes. From a set of 127 late-onset multiplex families screened for APOE, 43 (34%) families have at least one affected individual with no APOE-4 allele, suggesting an alternativemore » genetic etiology. Simulation studies indicated that additional loci could be identified through a genomic screen with a 10 cM sieve on a subset of 21 well documented, non-APOE-4 families. Given the uncertainties in the mode of inheritance, reliance on a single analytical method could result in a missed linkage. Therefore, we have developed a strategy of using multiple overlapping yet complementary methods to detect linkage. These include sib-pair analysis and affected-pedigree-member analysis, neither of which makes assumptions about mode of inheritance, and lod score analysis (using two predefined genetic models). In order for a marker to qualify for follow-up, it must fit at least two of three criteria. These are nominal P values of 0.05 or less for the non-parametric methods, and/or a lod score greater than 1.0. Adjacent markers each fulfilling a single criterion also warrant follow-up. To date, we have screened 61 markers on chromosomes 1, 2, 3, 18, 19, 21, and 22. One marker, D2S163, generated a lod score of 1.06 ({theta} = 0.15) and an APMT statistic of 3.68 (P < 0.001). This region is currently being investigated in more detail. Updated results of this region plus additional screening data will be presented.« less

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

PubMed Central

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

2015-01-01

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. PMID:24927667

Gene Addition Strategies for β-Thalassemia and Sickle Cell Anemia.

PubMed

Dong, Alisa C; Rivella, Stefano

2017-01-01

Beta-thalassemia and sickle cell anemia are two of the most common diseases related to the hemoglobin protein. In these diseases, the beta-globin gene is mutated, causing severe anemia and ineffective erythropoiesis. Patients can additionally present with a number of life-threatening co-morbidities, such as stroke or spontaneous fractures. Current treatment involves transfusion and iron chelation; allogeneic bone marrow transplant is the only curative option, but is limited by the availability of matching donors and graft-versus-host disease. As these two diseases are monogenic diseases, they make an attractive setting for gene therapy. Gene therapy aims to correct the mutated beta-globin gene or add back a functional copy of beta- or gamma-globin. Initial gene therapy work was done with oncoretroviral vectors, but has since shifted to lentiviral vectors. Currently, there are a few clinical trials underway to test the curative potential of some of these lentiviral vectors. This review will highlight the work done thus far, and present the challenges still facing gene therapy, such as genome toxicity concerns and achieving sufficient transgene expression to cure those with the most severe forms of thalassemia.

Time Course-Dependent Methanogenic Crude Oil Biodegradation: Dynamics of Fumarate Addition Metabolites, Biodegradative Genes, and Microbial Community Composition

PubMed Central

Toth, Courtney R. A.; Gieg, Lisa M.

2018-01-01

Biodegradation of crude oil in subsurface petroleum reservoirs has adversely impacted most of the world's oil, converting this resource to heavier forms that are of lower quality and more challenging to recover. Oil degradation in deep reservoir environments has been attributed to methanogenesis over geological time, yet our understanding of the processes and organisms mediating oil transformation in the absence of electron acceptors remains incomplete. Here, we sought to identify hydrocarbon activation mechanisms and reservoir-associated microorganisms that may have helped shape the formation of biodegraded oil by incubating oilfield produced water in the presence of light (°API = 32) or heavy crude oil (°API = 16). Over the course of 17 months, we conducted routine analytical (GC, GC-MS) and molecular (PCR/qPCR of assA and bssA genes, 16S rRNA gene sequencing) surveys to assess microbial community composition and activity changes over time. Over the incubation period, we detected the formation of transient hydrocarbon metabolites indicative of alkane and alkylbenzene addition to fumarate, corresponding with increases in methane production and fumarate addition gene abundance. Chemical and gene-based evidence of hydrocarbon biodegradation under methanogenic conditions was supported by the enrichment of hydrocarbon fermenters known to catalyze fumarate addition reactions (e.g., Desulfotomaculum, Smithella), along with syntrophic bacteria (Syntrophus), methanogenic archaea, and several candidate phyla (e.g., “Atribacteria”, “Cloacimonetes”). Our results reveal that fumarate addition is a possible mechanism for catalyzing the methanogenic biodegradation of susceptible saturates and aromatic hydrocarbons in crude oil, and we propose the roles of community members and candidate phyla in our cultures that may be involved in hydrocarbon transformation to methane in crude oil systems. PMID:29354103

Identification of potentially hazardous human gene products in GMO risk assessment.

PubMed

Bergmans, Hans; Logie, Colin; Van Maanen, Kees; Hermsen, Harm; Meredyth, Michelle; Van Der Vlugt, Cécile

2008-01-01

Genetically modified organisms (GMOs), e.g. viral vectors, could threaten the environment if by their release they spread hazardous gene products. Even in contained use, to prevent adverse consequences, viral vectors carrying genes from mammals or humans should be especially scrutinized as to whether gene products that they synthesize could be hazardous in their new context. Examples of such potentially hazardous gene products (PHGPs) are: protein toxins, products of dominant alleles that have a role in hereditary diseases, gene products and sequences involved in genome rearrangements, gene products involved in immunomodulation or with an endocrine function, gene products involved in apoptosis, activated proto-oncogenes. For contained use of a GMO that carries a construct encoding a PHGP, the precautionary principle dictates that safety measures should be applied on a "worst case" basis, until the risks of the specific case have been assessed. The potential hazard of cloned genes can be estimated before empirical data on the actual GMO become available. Preliminary data may be used to focus hazard identification and risk assessment. Both predictive and empirical data may also help to identify what further information is needed to assess the risk of the GMO. A two-step approach, whereby a PHGP is evaluated for its conceptual dangers, then checked by data bank searches, is delineated here.

Genome-Wide Screens Reveal New Gene Products That Influence Genetic Competence in Streptococcus mutans

PubMed Central

O'Brien, Greg; Maricic, Natalie; Kesterson, Alexandria; Grace, Megan

2017-01-01

ABSTRACT A network of genes and at least two peptide signaling molecules tightly control when Streptococcus mutans becomes competent to take up DNA from its environment. Widespread changes in the expression of genes occur when S. mutans is presented with competence signal peptides in vitro, including the increased production of the alternative sigma factor, ComX, which activates late competence genes. Still, the way that gene products that are regulated by competence peptides influence DNA uptake and cellular physiology are not well understood. Here, we developed and employed comprehensive transposon mutagenesis of the S. mutans genome, with a screen to identify mutants that aberrantly expressed comX, coupled with transposon sequencing (Tn-seq) to gain a more thorough understanding of the factors modulating comX expression and progression to the competent state. The screens effectively identified genes known to affect competence, e.g., comR, comS, comD, comE, cipB, clpX, rcrR, and ciaH, but disclosed an additional 20 genes that were not previously competence associated. The competence phenotypes of mutants were characterized, including by fluorescence microscopy to determine at which stage the mutants were impaired for comX activation. Among the novel genes studied were those implicated in cell division, the sensing of cell envelope stress, cell envelope biogenesis, and RNA stability. Our results provide a platform for determining the specific chemical and physical cues that are required for genetic competence in S. mutans, while highlighting the effectiveness of using Tn-seq in S. mutans to discover and study novel biological processes. IMPORTANCE Streptococcus mutans acquires DNA from its environment by becoming genetically competent, a physiologic state triggered by cell-cell communication using secreted peptides. Competence is important for acquiring novel genetic traits and has a strong influence on the expression of virulence-associated traits of S. mutans

Genome-wide screens reveal new gene products that influence genetic competence in Streptococcus mutans.

PubMed

Shields, Robert C; O'Brien, Greg; Maricic, Natalie; Kesterson, Alexandria; Grace, Megan; Hagen, Stephen J; Burne, Robert A

2017-11-06

A network of genes and at least two peptide signaling molecules tightly control when Streptococcus mutans becomes competent to take up DNA from its environment. Widespread changes in the expression of genes occur when S. mutans is presented with competence signal peptides in vitro , including increased production of the alternative sigma factor, ComX, which activates late competence genes. Still, the way that gene products that are regulated by competence peptides influence DNA uptake and cellular physiology are not well understood. Here, we developed and employed comprehensive transposon mutagenesis of the S. mutans genome with a screen to identify mutants that aberrantly expressed comX , coupled with transposon sequencing (Tn-seq) to gain a more thorough understanding of the factors modulating comX expression and progression to the competent state. The screens effectively identified genes known to affect competence, e.g. comR , comS , comD , comE , cipB , clpX , rcrR , ciaH , but disclosed an additional 20 genes that were not previously competence-associated. The competence phenotypes of mutants were characterized, including using fluorescence microscopy to determine at which stage the mutants were impaired for comX activation. Among the novel genes studied were those implicated in cell division, sensing of cell envelope stress, cell envelope biogenesis, and RNA stability. Our results provide a platform for determining the specific chemical and physical cues that are required for genetic competence in S. mutans , while highlighting the effectiveness of using Tn-seq in S. mutans to discover and study novel biological processes. IMPORTANCE Streptococcus mutans acquires DNA from its environment by becoming genetically competent, a physiologic state triggered by cell-cell communication using secreted peptides. Competence is important for acquiring novel genetic traits and has a strong influence on the expression of virulence-associated traits of S. mutans Here, we

Random transposon mutagenesis of the Saccharopolyspora erythraea genome reveals additional genes influencing erythromycin biosynthesis

PubMed Central

Fedashchin, Andrij; Cernota, William H.; Gonzalez, Melissa C.; Leach, Benjamin I.; Kwan, Noelle; Wesley, Roy K.; Weber, J. Mark

2015-01-01

A single cycle of strain improvement was performed in Saccharopolyspora erythraea mutB and 15 genotypes influencing erythromycin production were found. Genotypes generated by transposon mutagenesis appeared in the screen at a frequency of ∼3%. Mutations affecting central metabolism and regulatory genes were found, as well as hydrolases, peptidases, glycosyl transferases and unknown genes. Only one mutant retained high erythromycin production when scaled-up from micro-agar plug fermentations to shake flasks. This mutant had a knockout of the cwh1 gene (SACE_1598), encoding a cell-wall-associated hydrolase. The cwh1 knockout produced visible growth and morphological defects on solid medium. This study demonstrated that random transposon mutagenesis uncovers strain improvement-related genes potentially useful for strain engineering. PMID:26468041

Combining mouse mammary gland gene expression and comparative mapping for the identification of candidate genes for QTL of milk production traits in cattle

PubMed Central

Ron, Micha; Israeli, Galit; Seroussi, Eyal; Weller, Joel I; Gregg, Jeffrey P; Shani, Moshe; Medrano, Juan F

2007-01-01

Background Many studies have found segregating quantitative trait loci (QTL) for milk production traits in different dairy cattle populations. However, even for relatively large effects with a saturated marker map the confidence interval for QTL location by linkage analysis spans tens of map units, or hundreds of genes. Combining mapping and arraying has been suggested as an approach to identify candidate genes. Thus, gene expression analysis in the mammary gland of genes positioned in the confidence interval of the QTL can bridge the gap between fine mapping and quantitative trait nucleotide (QTN) determination. Results We hybridized Affymetrix microarray (MG-U74v2), containing 12,488 murine probes, with RNA derived from mammary gland of virgin, pregnant, lactating and involuting C57BL/6J mice in a total of nine biological replicates. We combined microarray data from two additional studies that used the same design in mice with a total of 75 biological replicates. The same filtering and normalization was applied to each microarray data using GeneSpring software. Analysis of variance identified 249 differentially expressed probe sets common to the three experiments along the four developmental stages of puberty, pregnancy, lactation and involution. 212 genes were assigned to their bovine map positions through comparative mapping, and thus form a list of candidate genes for previously identified QTLs for milk production traits. A total of 82 of the genes showed mammary gland-specific expression with at least 3-fold expression over the median representing all tissues tested in GeneAtlas. Conclusion This work presents a web tool for candidate genes for QTL (cgQTL) that allows navigation between the map of bovine milk production QTL, potential candidate genes and their level of expression in mammary gland arrays and in GeneAtlas. Three out of four confirmed genes that affect QTL in livestock (ABCG2, DGAT1, GDF8, IGF2) were over expressed in the target organ. Thus, cg

Detection of allergenic additives in processed meat products.

PubMed

Spychaj, Anita; Pospiech, Edward; Iwańska, Ewa; Montowska, Magdalena

2018-04-19

At present, allergic response to food components is an increasing problem all over the world. Therefore it is important to protect people vulnerable to food allergens against accidental and unintended consumption of products containing allergic ingredients. The meat industry commonly uses various allergic additives in production of processed products, i.e. legumes (soy, peas, beans), milk and egg preparations, cereals containing gluten (wheat, rye, barley and oats), and spices (celery and mustard). These meat additives have specific technological properties which help to create a texture, flavour profile or affect the nutritional value, although some of them, such as soy, mustard, milk and egg white proteins can cause severe allergic reactions. The aim of this paper is to discuss the application of various recently established methods of detection of allergenic additives in processed meat products, for instance cold cuts and sausages. The new methods are based mainly on protein, DNA and also isoflavones or phytic acid analysis. The article also characterises the latest trends in the development of research on the methods which would enable quick and reliable identification of targeted allergens in meat products. This article is protected by copyright. All rights reserved.

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

PubMed

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.

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

PubMed

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.

Production and pathogenicity of hepatitis C virus core gene products

PubMed Central

Li, Hui-Chun; Ma, Hsin-Chieh; Yang, Chee-Hing; Lo, Shih-Yen

2014-01-01

Hepatitis C virus (HCV) is a major cause of chronic liver diseases, including steatosis, cirrhosis and hepatocellular carcinoma, and its infection is also associated with insulin resistance and type 2 diabetes mellitus. HCV, belonging to the Flaviviridae family, is a small enveloped virus whose positive-stranded RNA genome encoding a polyprotein. The HCV core protein is cleaved first at residue 191 by the host signal peptidase and further cleaved by the host signal peptide peptidase at about residue 177 to generate the mature core protein (a.a. 1-177) and the cleaved peptide (a.a. 178-191). Core protein could induce insulin resistance, steatosis and even hepatocellular carcinoma through various mechanisms. The peptide (a.a. 178-191) may play a role in the immune response. The polymorphism of this peptide is associated with the cellular lipid drop accumulation, contributing to steatosis development. In addition to the conventional open reading frame (ORF), in the +1 frame, an ORF overlaps with the core protein-coding sequence and encodes the alternative reading frame proteins (ARFP or core+1). ARFP/core+1/F protein could enhance hepatocyte growth and may regulate iron metabolism. In this review, we briefly summarized the current knowledge regarding the production of different core gene products and their roles in viral pathogenesis. PMID:24966583

Epstein-Barr Virus BKRF4 Gene Product Is Required for Efficient Progeny Production.

PubMed

Masud, H M Abdullah Al; Watanabe, Takahiro; Yoshida, Masahiro; Sato, Yoshitaka; Goshima, Fumi; Kimura, Hiroshi; Murata, Takayuki

2017-12-01

Epstein-Barr virus (EBV), a member of human gammaherpesvirus, infects mainly B cells. EBV has two alternative life cycles, latent and lytic, and is reactivated occasionally from the latent stage to the lytic cycle. To combat EBV-associated disorders, understanding the molecular mechanisms of the EBV lytic replication cycle is also important. Here, we focused on an EBV lytic gene, BKRF4. Using our anti-BKRF4 antibody, we revealed that the BKRF4 gene product is expressed during the lytic cycle with late kinetics. To characterize the role of BKRF4, we constructed BKRF4-knockout mutants using the bacterial artificial chromosome (BAC) and CRISPR/Cas9 systems. Although disruption of the BKRF4 gene had almost no effect on viral protein expression and DNA synthesis, it significantly decreased progeny virion levels in HEK293 and Akata cells. Furthermore, we show that BKRF4 is involved not only in production of progeny virions but also in increasing the infectivity of the virus particles. Immunoprecipitation assays revealed that BKRF4 interacted with a virion protein, BGLF2. We showed that the C-terminal region of BKRF4 was critical for this interaction and for efficient progeny production. Immunofluorescence analysis revealed that BKRF4 partially colocalized with BGLF2 in the nucleus and perinuclear region. Finally, we showed that BKRF4 is a phosphorylated, possible tegument protein and that the EBV protein kinase BGLF4 may be important for this phosphorylation. Taken together, our data suggest that BKRF4 is involved in the production of infectious virions. IMPORTANCE Although the latent genes of EBV have been studied extensively, the lytic genes are less well characterized. This study focused on one such lytic gene, BKRF4, which is conserved only among gammaherpesviruses (ORF45 of Kaposi's sarcoma-associated herpesvirus or murine herpesvirus 68). After preparing the BKRF4 knockout virus using B95-8 EBV-BAC, we demonstrated that the BKRF4 gene was involved in infectious

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

PubMed

Mo, Delin; Zhu, Zhengmao; te Pas, Marinus F W; Li, Xinyun; Yang, Shulin; Wang, Heng; Wang, Huanling; Li, Kui

2008-06-30

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. 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 < 0.05). 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.

Potential hazards due to food additives in oral hygiene products.

PubMed

Tuncer Budanur, Damla; Yas, Murat Cengizhan; Sepet, Elif

2016-01-01

Food additives used to preserve flavor or to enhance the taste and appearance of foods are also available in oral hygiene products. The aim of this review is to provide information concerning food additives in oral hygiene products and their adverse effects. A great many of food additives in oral hygiene products are potential allergens and they may lead to allergic reactions such as urticaria, contact dermatitis, rhinitis, and angioedema. Dental practitioners, as well as health care providers, must be aware of the possibility of allergic reactions due to food additives in oral hygiene products. Proper dosage levels, delivery vehicles, frequency, potential benefits, and adverse effects of oral health products should be explained completely to the patients. There is a necessity to raise the awareness among dental professionals on this subject and to develop a data gathering system for possible adverse reactions.

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

DOEpatents

Desprez, Pierre-Yves; Campisi, Judith

2014-08-19

A method for treatment of breast cancer and other types of cancer. The method comprises targeting and modulating Id-1 gene expression, if any, for the Id-1 gene, or gene products in breast or other epithelial cancers in a patient by delivering products that modulate Id-1 gene expression. When expressed, Id-1 gene is a prognostic indicator that cancer cells are invasive and metastatic.

Addition products of alpha-tocopherol with lipid-derived free radicals.

PubMed

Yamauchi, Ryo

2007-01-01

The addition products of alpha-tocopherol with lipid-derived free radicals have been reviewed. Free radical scavenging reactions of alpha-tocopherol take place via the alpha-tocopheroxyl radical as an intermediate. If a suitable free radical is present, an addition product can be formed from the coupling of the free radical with the alpha-tocopheroxyl radical. The addition products of alpha-tocopherol with lipid-peroxyl radicals are 8a-(lipid-dioxy)-alpha-tocopherones, which are hydrolyzed to alpha-tocopherylquinone. On the other hand, the carbon-centered radicals of lipids prefer to react with the phenoxyl radical of alpha-tocopherol to form 6-O-lipid-alpha-tocopherol under anaerobic conditions. The addition products of alpha-tocopherol with peroxyl radicals (epoxylinoleoyl-peroxyl radicals) produced from cholesteryl ester and phosphatidylcholine were detected in the peroxidized human plasma using a high-sensitive HPLC procedure with postcolumn reduction and electrochemical detection. Thus, the formation of these addition products provides us with much information on the antioxidant function of vitamin E in biological systems.

Productive performance of the dairy cattle Girolando breed mediated by the fat-related genes DGAT1 and LEP and their polymorphisms.

PubMed

Cardoso, S R; Queiroz, L B; Goulart, V Alonso; Mourão, G B; Benedetti, E; Goulart, L R

2011-12-01

Candidate genes have been associated with milk production in bovines, such as the diacylglycerol O-acyltransferase 1 (DGAT1) and leptin (LEP); however, they have not been simultaneously investigated nor have been evaluated in the Brazilian Girolando breed (Gir×Holstein, backcrossed to Holstein). Our aim was to determine the influence of fat-related genes, DGAT1 and LEP, and their polymorphisms on performance traits of milk production in the Girolando breed. Results indicated that the K allele of the DGAT1 gene showed a significant association with total and average daily milk production with additive effect. The LEP gene showed that the A allele and its homozygote are highly prevalent and almost fixed in this population and may have been favorably selected during backcrossing for the origin of this breed. The important impact of the K allele of the DGAT1 gene on milk production corroborates the initiative of performing marker-assisted selections with this gene in breeding programs of the Girolando breed. Copyright © 2011 Elsevier Ltd. All rights reserved.

Molecular characterization of an unauthorized genetically modified Bacillus subtilis production strain identified in a vitamin B2 feed additive.

PubMed

Paracchini, Valentina; Petrillo, Mauro; Reiting, Ralf; Angers-Loustau, Alexandre; Wahler, Daniela; Stolz, Andrea; Schönig, Birgit; Matthies, Anastasia; Bendiek, Joachim; Meinel, Dominik M; Pecoraro, Sven; Busch, Ulrich; Patak, Alex; Kreysa, Joachim; Grohmann, Lutz

2017-09-01

Many food and feed additives result from fermentation of genetically modified (GM) microorganisms. For vitamin B2 (riboflavin), GM Bacillus subtilis production strains have been developed and are often used. The presence of neither the GM strain nor its recombinant DNA is allowed for fermentation products placed on the EU market as food or feed additive. A vitamin B 2 product (80% feed grade) imported from China was analysed. Viable B. subtilis cells were identified and DNAs of two bacterial isolates (LHL and LGL) were subjected to three whole genome sequencing (WGS) runs with different devices (MiSeq, 454 or HiSeq system). WGS data revealed the integration of a chloramphenicol resistance gene, the deletion of the endogenous riboflavin (rib) operon and presence of four putative plasmids harbouring rib operons. Event- and construct-specific real-time PCR methods for detection of the GM strain and its putative plasmids in food and feed products have been developed. Copyright © 2017 Elsevier Ltd. All rights reserved.

15 CFR 1180.6 - Production of additional copies.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 15 Commerce and Foreign Trade 3 2010-01-01 2010-01-01 false Production of additional copies. 1180... (Continued) TECHNOLOGY ADMINISTRATION, DEPARTMENT OF COMMERCE TRANSFER BY FEDERAL AGENCIES OF SCIENTIFIC, TECHNICAL AND ENGINEERING INFORMATION TO THE NATIONAL TECHNICAL INFORMATION SERVICE § 1180.6 Production of...

A transversal approach to predict gene product networks from ontology-based similarity

PubMed Central

Chabalier, Julie; Mosser, Jean; Burgun, Anita

2007-01-01

Background Interpretation of transcriptomic data is usually made through a "standard" approach which consists in clustering the genes according to their expression patterns and exploiting Gene Ontology (GO) annotations within each expression cluster. This approach makes it difficult to underline functional relationships between gene products that belong to different expression clusters. To address this issue, we propose a transversal analysis that aims to predict functional networks based on a combination of GO processes and data expression. Results The transversal approach presented in this paper consists in computing the semantic similarity between gene products in a Vector Space Model. Through a weighting scheme over the annotations, we take into account the representativity of the terms that annotate a gene product. Comparing annotation vectors results in a matrix of gene product similarities. Combined with expression data, the matrix is displayed as a set of functional gene networks. The transversal approach was applied to 186 genes related to the enterocyte differentiation stages. This approach resulted in 18 functional networks proved to be biologically relevant. These results were compared with those obtained through a standard approach and with an approach based on information content similarity. Conclusion Complementary to the standard approach, the transversal approach offers new insight into the cellular mechanisms and reveals new research hypotheses by combining gene product networks based on semantic similarity, and data expression. PMID:17605807

Towards Production of Additive Manufacturing Grade Metallic Powders on the Battlefield

DTIC Science & Technology

2017-10-01

ARL-RP-0618 ● OCT 2017 US Army Research Laboratory Towards Production of Additive Manufacturing Grade Metallic Powders on the...Research Laboratory Towards Production of Additive Manufacturing Grade Metallic Powders on the Battlefield by Marc Pepi Weapons and...REPORT TYPE Reprint 3. DATES COVERED (From - To) June 2016–June 2017 4. TITLE AND SUBTITLE Towards Production of Additive Manufacturing Grade

Identification of flavour additives in tobacco products to develop a flavour library

PubMed Central

Krüsemann, Erna JZ; Visser, Wouter F; Cremers, Johannes WJM; Pennings, Jeroen LA; Talhout, Reinskje

2018-01-01

Objectives This study combines chemical analysis and flavour descriptions of flavour additives used in tobacco products, and provides a starting point to build an extensive library of flavour components, useful for product surveillance. Methods Headspace gas chromatography-mass spectrometry (GC-MS) was used to compare 22 commercially available tobacco products (cigarettes and roll-your-own) expected to have a characterising flavour and 6 commercially available products not expected to have a characterising flavour with 5 reference products (natural tobacco leaves and research cigarettes containing no flavour additives). The flavour components naturally present in the reference products were excluded from components present in commercially available products containing flavour additives. A description of the remaining flavour additives was used for categorisation. Results GC-MS measurements of the 33 tobacco products resulted in an overview of 186 chemical compounds. Of these, 144 were solely present in commercially available products. These 144 flavour additives were described using 62 different flavour descriptors extracted from flavour databases, which were categorised into eight groups largely based on the definition of characterising flavours from the European Tobacco Product Directive: fruit, spice, herb, alcohol, menthol, sweet, floral and miscellaneous. Conclusions We developed a method to identify and describe flavour additives in tobacco products. Flavour additives consist of single flavour compounds or mixtures of multiple flavour compounds, and different combinations of flavour compounds can cause a certain flavour. A flavour library helps to detect flavour additives that are characteristic for a certain flavour, and thus can be useful for regulation of flavours in tobacco and related products. PMID:28190004

Regulation of Cell and Gene Therapy Medicinal Products in Taiwan.

PubMed

Lin, Yi-Chu; Wang, Po-Yu; Tsai, Shih-Chih; Lin, Chien-Liang; Tai, Hsuen-Yung; Lo, Chi-Fang; Wu, Shiow-Ing; Chiang, Yu-Mei; Liu, Li-Ling

2015-01-01

Owing to the rapid and mature development of emerging biotechnology in the fields of cell culture, cell preservation, and recombinant DNA technology, more and more cell or gene medicinal therapy products have been approved for marketing, to treat serious diseases which have been challenging to treat with current medical practice or medicine. This chapter will briefly introduce the Taiwan Food and Drug Administration (TFDA) and elaborate regulation of cell and gene therapy medicinal products in Taiwan, including regulatory history evolution, current regulatory framework, application and review procedures, and relevant jurisdictional issues. Under the promise of quality, safety, and efficacy of medicinal products, it is expected the regulation and environment will be more flexible, streamlining the process of the marketing approval of new emerging cell or gene therapy medicinal products and providing diverse treatment options for physicians and patients.

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

PubMed

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.

Additive genetic variation and evolvability of a multivariate trait can be increased by epistatic gene action.

PubMed

Griswold, Cortland K

2015-12-21

Epistatic gene action occurs when mutations or alleles interact to produce a phenotype. Theoretically and empirically it is of interest to know whether gene interactions can facilitate the evolution of diversity. In this paper, we explore how epistatic gene action affects the additive genetic component or heritable component of multivariate trait variation, as well as how epistatic gene action affects the evolvability of multivariate traits. The analysis involves a sexually reproducing and recombining population. Our results indicate that under stabilizing selection conditions a population with a mixed additive and epistatic genetic architecture can have greater multivariate additive genetic variation and evolvability than a population with a purely additive genetic architecture. That greater multivariate additive genetic variation can occur with epistasis is in contrast to previous theory that indicated univariate additive genetic variation is decreased with epistasis under stabilizing selection conditions. In a multivariate setting, epistasis leads to less relative covariance among individuals in their genotypic, as well as their breeding values, which facilitates the maintenance of additive genetic variation and increases a population׳s evolvability. Our analysis involves linking the combinatorial nature of epistatic genetic effects to the ancestral graph structure of a population to provide insight into the consequences of epistasis on multivariate trait variation and evolution. Copyright © 2015 Elsevier Ltd. All rights reserved.

Genetic resources for advanced biofuel production described with the Gene Ontology.

PubMed

Torto-Alalibo, Trudy; Purwantini, Endang; Lomax, Jane; Setubal, João C; Mukhopadhyay, Biswarup; Tyler, Brett M

2014-01-01

Dramatic increases in research in the area of microbial biofuel production coupled with high-throughput data generation on bioenergy-related microbes has led to a deluge of information in the scientific literature and in databases. Consolidating this information and making it easily accessible requires a unified vocabulary. The Gene Ontology (GO) fulfills that requirement, as it is a well-developed structured vocabulary that describes the activities and locations of gene products in a consistent manner across all kingdoms of life. The Microbial ENergy processes Gene Ontology () project is extending the GO to include new terms to describe microbial processes of interest to bioenergy production. Our effort has added over 600 bioenergy related terms to the Gene Ontology. These terms will aid in the comprehensive annotation of gene products from diverse energy-related microbial genomes. An area of microbial energy research that has received a lot of attention is microbial production of advanced biofuels. These include alcohols such as butanol, isopropanol, isobutanol, and fuels derived from fatty acids, isoprenoids, and polyhydroxyalkanoates. These fuels are superior to first generation biofuels (ethanol and biodiesel esterified from vegetable oil or animal fat), can be generated from non-food feedstock sources, can be used as supplements or substitutes for gasoline, diesel and jet fuels, and can be stored and distributed using existing infrastructure. Here we review the roles of genes associated with synthesis of advanced biofuels, and at the same time introduce the use of the GO to describe the functions of these genes in a standardized way.

Genetic resources for advanced biofuel production described with the Gene Ontology

PubMed Central

Torto-Alalibo, Trudy; Purwantini, Endang; Lomax, Jane; Setubal, João C.; Mukhopadhyay, Biswarup; Tyler, Brett M.

2014-01-01

Dramatic increases in research in the area of microbial biofuel production coupled with high-throughput data generation on bioenergy-related microbes has led to a deluge of information in the scientific literature and in databases. Consolidating this information and making it easily accessible requires a unified vocabulary. The Gene Ontology (GO) fulfills that requirement, as it is a well-developed structured vocabulary that describes the activities and locations of gene products in a consistent manner across all kingdoms of life. The Microbial ENergy processes Gene Ontology () project is extending the GO to include new terms to describe microbial processes of interest to bioenergy production. Our effort has added over 600 bioenergy related terms to the Gene Ontology. These terms will aid in the comprehensive annotation of gene products from diverse energy-related microbial genomes. An area of microbial energy research that has received a lot of attention is microbial production of advanced biofuels. These include alcohols such as butanol, isopropanol, isobutanol, and fuels derived from fatty acids, isoprenoids, and polyhydroxyalkanoates. These fuels are superior to first generation biofuels (ethanol and biodiesel esterified from vegetable oil or animal fat), can be generated from non-food feedstock sources, can be used as supplements or substitutes for gasoline, diesel and jet fuels, and can be stored and distributed using existing infrastructure. Here we review the roles of genes associated with synthesis of advanced biofuels, and at the same time introduce the use of the GO to describe the functions of these genes in a standardized way. PMID:25346727

Genetic resources for advanced biofuel production described with the Gene Ontology

DOE PAGES

Torto-Alalibo, Trudy; Purwantini, Endang; Lomax, Jane; ...

2014-10-10

Dramatic increases in research in the area of microbial biofuel production coupled with high-throughput data generation on bioenergy-related microbes has led to a deluge of information in the scientific literature and in databases. Consolidating this information and making it easily accessible requires a unified vocabulary.The Gene Ontology (GO) fulfills that requirement, as it is a well-developed structured vocabulary that describes the activities and locations of gene products in a consistent manner across all kingdoms of life. The Microbial ENergy processes Gene Ontology (http://www.mengo.biochem.vt.edu) project is extending the GO to include new terms to describe microbial processes of interest to bioenergymore » production. Our effort has added over 600 bioenergy related terms to the Gene Ontology. These terms will aid in the comprehensive annotation of gene products from diverse energy-related microbial genomes. An area of microbial energy research that has received a lot of attention is microbial production of advanced biofuels. These include alcohols such as butanol, isopropanol, isobutanol, and fuels derived from fatty acids, isoprenoids, and polyhydroxyalkanoates. These fuels are superior to first generation biofuels (ethanol and biodiesel esterified from vegetable oil or animal fat), can be generated from non-food feedstock sources, can be used as supplements or substitutes for gasoline, diesel and jet fuels, and can be stored and distributed using existing infrastructure. We review the roles of genes associated with synthesis of advanced biofuels, and at the same time introduce the use of the GO to describe the functions of these genes in a standardized way.« less

Genetic resources for advanced biofuel production described with the Gene Ontology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Torto-Alalibo, Trudy; Purwantini, Endang; Lomax, Jane

Dramatic increases in research in the area of microbial biofuel production coupled with high-throughput data generation on bioenergy-related microbes has led to a deluge of information in the scientific literature and in databases. Consolidating this information and making it easily accessible requires a unified vocabulary.The Gene Ontology (GO) fulfills that requirement, as it is a well-developed structured vocabulary that describes the activities and locations of gene products in a consistent manner across all kingdoms of life. The Microbial ENergy processes Gene Ontology (http://www.mengo.biochem.vt.edu) project is extending the GO to include new terms to describe microbial processes of interest to bioenergymore » production. Our effort has added over 600 bioenergy related terms to the Gene Ontology. These terms will aid in the comprehensive annotation of gene products from diverse energy-related microbial genomes. An area of microbial energy research that has received a lot of attention is microbial production of advanced biofuels. These include alcohols such as butanol, isopropanol, isobutanol, and fuels derived from fatty acids, isoprenoids, and polyhydroxyalkanoates. These fuels are superior to first generation biofuels (ethanol and biodiesel esterified from vegetable oil or animal fat), can be generated from non-food feedstock sources, can be used as supplements or substitutes for gasoline, diesel and jet fuels, and can be stored and distributed using existing infrastructure. We review the roles of genes associated with synthesis of advanced biofuels, and at the same time introduce the use of the GO to describe the functions of these genes in a standardized way.« less

An Analysis Of Additive Manufacturing Production Problems And Solutions

DTIC Science & Technology

2016-12-01

democratization of manufacturing (Hornick, 2015). AM has three distinct advantages over subtractive manufacturing : product customization, design flexibility...58 develops software and other technology solutions for the design , analysis, testing, manufacture , and validation of products (Diane Ryan, personal...NAVAL POSTGRADUATE SCHOOL MONTEREY, CALIFORNIA MBA PROFESSIONAL REPORT AN ANALYSIS OF ADDITIVE MANUFACTURING PRODUCTION

Identification of flavour additives in tobacco products to develop a flavour library.

PubMed

Krüsemann, Erna Jz; Visser, Wouter F; Cremers, Johannes Wjm; Pennings, Jeroen LA; Talhout, Reinskje

2018-01-01

This study combines chemical analysis and flavour descriptions of flavour additives used in tobacco products, and provides a starting point to build an extensive library of flavour components, useful for product surveillance. Headspace gas chromatography-mass spectrometry (GC-MS) was used to compare 22 commercially available tobacco products (cigarettes and roll-your-own) expected to have a characterising flavour and 6 commercially available products not expected to have a characterising flavour with 5 reference products (natural tobacco leaves and research cigarettes containing no flavour additives). The flavour components naturally present in the reference products were excluded from components present in commercially available products containing flavour additives. A description of the remaining flavour additives was used for categorisation. GC-MS measurements of the 33 tobacco products resulted in an overview of 186 chemical compounds. Of these, 144 were solely present in commercially available products. These 144 flavour additives were described using 62 different flavour descriptors extracted from flavour databases, which were categorised into eight groups largely based on the definition of characterising flavours from the European Tobacco Product Directive: fruit, spice, herb, alcohol, menthol, sweet, floral and miscellaneous. We developed a method to identify and describe flavour additives in tobacco products. Flavour additives consist of single flavour compounds or mixtures of multiple flavour compounds, and different combinations of flavour compounds can cause a certain flavour. A flavour library helps to detect flavour additives that are characteristic for a certain flavour, and thus can be useful for regulation of flavours in tobacco and related products. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Inhibitory effects of food additives derived from polyphenols on staphylococcal enterotoxin A production and biofilm formation by Staphylococcus aureus.

PubMed

Shimamura, Yuko; Hirai, Chikako; Sugiyama, Yuka; Shibata, Masaharu; Ozaki, Junya; Murata, Masatsune; Ohashi, Norio; Masuda, Shuichi

2017-12-01

In this study, we examined the inhibitory effects of 14 food additives derived from polyphenol samples on staphylococcal enterotoxin A (SEA) production and biofilm formation by Staphylococcus aureus. Tannic acid AL (TA), Purephenon 50 W (PP) and Polyphenon 70A (POP) at 0.25 mg/mL and Gravinol®-N (GN), Blackcurrant polyphenol AC10 (BP), and Resveratrol-P5 (RT) at 1.0 mg/mL significantly decreased SEA production by S. aureus C-29 (p < 0.05). TA, GN, BP, and RT significantly inhibited the expression of the sea gene in S. aureus C-29 (p < 0.05), while suppression attempts by PP and POP proved unsuccessful. After result analysis, it can be derived that TA, GN, BP, and RT inhibit the production of SEA. Of the six samples, each one significantly inhibited biofilm formation (p < 0.05). Food additives derived from polyphenols have viability to be used as a means to inhibit the enterotoxin production and control the biofilm formation of foodborne pathogens.

78 FR 53486 - Addition of Round-Trip Mailer Product to the Competitive Product List

Federal Register 2010, 2011, 2012, 2013, 2014

2013-08-29

... POSTAL SERVICE Addition of Round-Trip Mailer Product to the Competitive Product List AGENCY... request with the Postal Regulatory Commission to add a product called ``Round-Trip Mailer'' to the...] filed with the Postal Regulatory Commission (Commission) a request to add a ``Round-Trip Mailer...

Functional Analysis of the Gene Cluster Involved in Production of the Bacteriocin Circularin A by Clostridium beijerinckii ATCC 25752

PubMed Central

Kemperman, Robèr; Jonker, Marnix; Nauta, Arjen; Kuipers, Oscar P.; Kok, Jan

2003-01-01

A region of 12 kb flanking the structural gene of the cyclic antibacterial peptide circularin A of Clostridium beijerinckii ATCC 25752 was sequenced, and the putative proteins involved in the production and secretion of circularin A were identified. The genes are tightly organized in overlapping open reading frames. Heterologous expression of circularin A in Enterococcus faecalis was achieved, and five genes were identified as minimally required for bacteriocin production and secretion. Two of the putative proteins, CirB and CirC, are predicted to contain membrane-spanning domains, while CirD contains a highly conserved ATP-binding domain. Together with CirB and CirC, this ATP-binding protein is involved in the production of circularin A. The fifth gene, cirE, confers immunity towards circularin A when expressed in either Lactococcus lactis or E. faecalis and is needed in order to allow the bacteria to produce bacteriocin. Additional resistance against circularin A is conferred by the activity of the putative transporter consisting of CirB and CirD. PMID:14532033

Reanalysis of RNA-Sequencing Data Reveals Several Additional Fusion Genes with Multiple Isoforms

PubMed Central

Kangaspeska, Sara; Hultsch, Susanne; Edgren, Henrik; Nicorici, Daniel; Murumägi, Astrid; Kallioniemi, Olli

2012-01-01

RNA-sequencing and tailored bioinformatic methodologies have paved the way for identification of expressed fusion genes from the chaotic genomes of solid tumors. We have recently successfully exploited RNA-sequencing for the discovery of 24 novel fusion genes in breast cancer. Here, we demonstrate the importance of continuous optimization of the bioinformatic methodology for this purpose, and report the discovery and experimental validation of 13 additional fusion genes from the same samples. Integration of copy number profiling with the RNA-sequencing results revealed that the majority of the gene fusions were promoter-donating events that occurred at copy number transition points or involved high-level DNA-amplifications. Sequencing of genomic fusion break points confirmed that DNA-level rearrangements underlie selected fusion transcripts. Furthermore, a significant portion (>60%) of the fusion genes were alternatively spliced. This illustrates the importance of reanalyzing sequencing data as gene definitions change and bioinformatic methods improve, and highlights the previously unforeseen isoform diversity among fusion transcripts. PMID:23119097

Reanalysis of RNA-sequencing data reveals several additional fusion genes with multiple isoforms.

PubMed

Kangaspeska, Sara; Hultsch, Susanne; Edgren, Henrik; Nicorici, Daniel; Murumägi, Astrid; Kallioniemi, Olli

2012-01-01

RNA-sequencing and tailored bioinformatic methodologies have paved the way for identification of expressed fusion genes from the chaotic genomes of solid tumors. We have recently successfully exploited RNA-sequencing for the discovery of 24 novel fusion genes in breast cancer. Here, we demonstrate the importance of continuous optimization of the bioinformatic methodology for this purpose, and report the discovery and experimental validation of 13 additional fusion genes from the same samples. Integration of copy number profiling with the RNA-sequencing results revealed that the majority of the gene fusions were promoter-donating events that occurred at copy number transition points or involved high-level DNA-amplifications. Sequencing of genomic fusion break points confirmed that DNA-level rearrangements underlie selected fusion transcripts. Furthermore, a significant portion (>60%) of the fusion genes were alternatively spliced. This illustrates the importance of reanalyzing sequencing data as gene definitions change and bioinformatic methods improve, and highlights the previously unforeseen isoform diversity among fusion transcripts.

Complete sequence of a plasmid from a bovine methicillin-resistant Staphylococcus aureus harbouring a novel ica-like gene cluster in addition to antimicrobial and heavy metal resistance genes.

PubMed

Feßler, Andrea T; Zhao, Qin; Schoenfelder, Sonja; Kadlec, Kristina; Brenner Michael, Geovana; Wang, Yang; Ziebuhr, Wilma; Shen, Jianzhong; Schwarz, Stefan

2017-02-01

The multiresistance plasmid pAFS11, obtained from a bovine methicillin-resistant Staphylococcus aureus (MRSA) isolate, was completely sequenced and analysed for its structure and organisation. Moreover, the susceptibility to the heavy metals cadmium and copper was determined by broth macrodilution. The 49,189-bp plasmid harboured the apramycin resistance gene apmA, two copies of the macrolide/lincosamide/streptogramin B resistance gene erm(B) (both located on remnants of a truncated transposon Tn917), the kanamycin/neomycin resistance gene aadD, the tetracycline resistance gene tet(L) and the trimethoprim resistance gene dfrK. The latter three genes were part of a 7,284-bp segment which was bracketed by two copies of IS431. In addition, the cadmium resistance operon cadDX as well as the copper resistance genes copA and mco were located on the plasmid and mediated a reduced susceptibility to cadmium and copper. Moreover, a complete novel ica-like gene cluster of so far unknown genetic origin was detected on this plasmid. The ica-like gene cluster comprised four different genes whose products showed 64.4-76.9% homology to the Ica proteins known to be involved in biofilm formation of the S. aureus strains Mu50, Mu3 and N315. However, 96.2-99.4% homology was seen to proteins from S. sciuri NS1 indicating an S. sciuri origin. The finding of five different antibiotic resistance genes co-located on a plasmid with heavy metal resistance genes and an ica-like gene cluster is alarming. With the acquisition of this plasmid, antimicrobial multiresistance, heavy metal resistances and potential virulence properties may be co-selected and spread via a single horizontal gene transfer event. Copyright © 2016 Elsevier B.V. All rights reserved.

Discovery of single nucleotide polymorphisms in candidate genes associated with fertility and production traits in Holstein cattle

PubMed Central

2013-01-01

Background Identification of single nucleotide polymorphisms (SNPs) for specific genes involved in reproduction might improve reliability of genomic estimates for these low-heritability traits. Semen from 550 Holstein bulls of high (≥ 1.7; n = 288) or low (≤ −2; n = 262) daughter pregnancy rate (DPR) was genotyped for 434 candidate SNPs using the Sequenom MassARRAY® system. Three types of SNPs were evaluated: SNPs previously reported to be associated with reproductive traits or physically close to genetic markers for reproduction, SNPs in genes that are well known to be involved in reproductive processes, and SNPs in genes that are differentially expressed between physiological conditions in a variety of tissues associated in reproductive function. Eleven reproduction and production traits were analyzed. Results A total of 40 SNPs were associated (P < 0.05) with DPR. Among these were genes involved in the endocrine system, cell signaling, immune function and inhibition of apoptosis. A total of 10 genes were regulated by estradiol. In addition, 22 SNPs were associated with heifer conception rate, 33 with cow conception rate, 36 with productive life, 34 with net merit, 23 with milk yield, 19 with fat yield, 13 with fat percent, 19 with protein yield, 22 with protein percent, and 13 with somatic cell score. The allele substitution effect for SNPs associated with heifer conception rate, cow conception rate, productive life and net merit were in the same direction as for DPR. Allele substitution effects for several SNPs associated with production traits were in the opposite direction as DPR. Nonetheless, there were 29 SNPs associated with DPR that were not negatively associated with production traits. Conclusion SNPs in a total of 40 genes associated with DPR were identified as well as SNPs for other traits. It might be feasible to include these SNPs into genomic tests of reproduction and other traits. The genes associated with DPR are likely to be

Polyhydroxyalkanoate production from sucrose by Cupriavidus necator strains harboring csc genes from Escherichia coli W.

PubMed

Arikawa, Hisashi; Matsumoto, Keiji; Fujiki, Tetsuya

2017-10-01

Cupriavidus necator H16 is the most promising bacterium for industrial production of polyhydroxyalkanoates (PHAs) because of their remarkable ability to accumulate them in the cells. With genetic modifications, this bacterium can produce poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), which has better physical properties, as well as poly(3-hydroxybutyrate) (PHB) using plant oils and sugars as a carbon source. Considering production cost, sucrose is a very attractive raw material because it is inexpensive; however, this bacterium cannot assimilate sucrose. Here, we used the sucrose utilization (csc) genes of Escherichia coli W to generate C. necator strains that can assimilate sucrose. Especially, glucose-utilizing recombinant C. necator strains harboring the sucrose hydrolase gene (cscA) and sucrose permease gene (cscB) of E. coli W grew well on sucrose as a sole carbon source and accumulated PHB. In addition, strains introduced with a crotonyl-CoA reductase gene (ccr), ethylmalonyl-CoA decarboxylase gene (emd), and some other genetic modifications besides the csc genes and the glucose-utilizing mutations produced PHBHHx with a 3-hydroxyhexanoate (3HHx) content of maximum approximately 27 mol% from sucrose. Furthermore, when one of the PHBHHx-producing strains was cultured with sucrose solution in a fed-batch fermentation, PHBHHx with a 3HHx content of approximately 4 mol% was produced and reached 113 g/L for 65 h, which is approximately 1.5-fold higher than that produced using glucose solution.

Gene cluster conservation provides insight into cercosporin biosynthesis and extends production to the genus Colletotrichum.

PubMed

de Jonge, Ronnie; Ebert, Malaika K; Huitt-Roehl, Callie R; Pal, Paramita; Suttle, Jeffrey C; Spanner, Rebecca E; Neubauer, Jonathan D; Jurick, Wayne M; Stott, Karina A; Secor, Gary A; Thomma, Bart P H J; Van de Peer, Yves; Townsend, Craig A; Bolton, Melvin D

2018-06-12

Species in the genus Cercospora cause economically devastating diseases in sugar beet, maize, rice, soy bean, and other major food crops. Here, we sequenced the genome of the sugar beet pathogen Cercospora beticola and found it encodes 63 putative secondary metabolite gene clusters, including the cercosporin toxin biosynthesis ( CTB ) cluster. We show that the CTB gene cluster has experienced multiple duplications and horizontal transfers across a spectrum of plant pathogenic fungi, including the wide-host range Colletotrichum genus as well as the rice pathogen Magnaporthe oryzae Although cercosporin biosynthesis has been thought to rely on an eight-gene CTB cluster, our phylogenomic analysis revealed gene collinearity adjacent to the established cluster in all CTB cluster-harboring species. We demonstrate that the CTB cluster is larger than previously recognized and includes cercosporin facilitator protein, previously shown to be involved with cercosporin autoresistance, and four additional genes required for cercosporin biosynthesis, including the final pathway enzymes that install the unusual cercosporin methylenedioxy bridge. Lastly, we demonstrate production of cercosporin by Colletotrichum fioriniae , the first known cercosporin producer within this agriculturally important genus. Thus, our results provide insight into the intricate evolution and biology of a toxin critical to agriculture and broaden the production of cercosporin to another fungal genus containing many plant pathogens of important crops worldwide. Copyright © 2018 the Author(s). Published by PNAS.

Identification of Linkages between EDCs in Personal Care Products and Breast Cancer through Data Integration Combined with Gene Network Analysis.

PubMed

Jeong, Hyeri; Kim, Jongwoon; Kim, Youngjun

2017-09-30

Approximately 1000 chemicals have been reported to possibly have endocrine disrupting effects, some of which are used in consumer products, such as personal care products (PCPs) and cosmetics. We conducted data integration combined with gene network analysis to: (i) identify causal molecular mechanisms between endocrine disrupting chemicals (EDCs) used in PCPs and breast cancer; and (ii) screen candidate EDCs associated with breast cancer. Among EDCs used in PCPs, four EDCs having correlation with breast cancer were selected, and we curated 27 common interacting genes between those EDCs and breast cancer to perform the gene network analysis. Based on the gene network analysis, ESR1, TP53, NCOA1, AKT1, and BCL6 were found to be key genes to demonstrate the molecular mechanisms of EDCs in the development of breast cancer. Using GeneMANIA, we additionally predicted 20 genes which could interact with the 27 common genes. In total, 47 genes combining the common and predicted genes were functionally grouped with the gene ontology and KEGG pathway terms. With those genes, we finally screened candidate EDCs for their potential to increase breast cancer risk. This study highlights that our approach can provide insights to understand mechanisms of breast cancer and identify potential EDCs which are in association with breast cancer.

Identification of Linkages between EDCs in Personal Care Products and Breast Cancer through Data Integration Combined with Gene Network Analysis

PubMed Central

Kim, Jongwoon

2017-01-01

Approximately 1000 chemicals have been reported to possibly have endocrine disrupting effects, some of which are used in consumer products, such as personal care products (PCPs) and cosmetics. We conducted data integration combined with gene network analysis to: (i) identify causal molecular mechanisms between endocrine disrupting chemicals (EDCs) used in PCPs and breast cancer; and (ii) screen candidate EDCs associated with breast cancer. Among EDCs used in PCPs, four EDCs having correlation with breast cancer were selected, and we curated 27 common interacting genes between those EDCs and breast cancer to perform the gene network analysis. Based on the gene network analysis, ESR1, TP53, NCOA1, AKT1, and BCL6 were found to be key genes to demonstrate the molecular mechanisms of EDCs in the development of breast cancer. Using GeneMANIA, we additionally predicted 20 genes which could interact with the 27 common genes. In total, 47 genes combining the common and predicted genes were functionally grouped with the gene ontology and KEGG pathway terms. With those genes, we finally screened candidate EDCs for their potential to increase breast cancer risk. This study highlights that our approach can provide insights to understand mechanisms of breast cancer and identify potential EDCs which are in association with breast cancer. PMID:28973975

Form gene clustering method about pan-ethnic-group products based on emotional semantic

NASA Astrophysics Data System (ADS)

Chen, Dengkai; Ding, Jingjing; Gao, Minzhuo; Ma, Danping; Liu, Donghui

2016-09-01

The use of pan-ethnic-group products form knowledge primarily depends on a designer's subjective experience without user participation. The majority of studies primarily focus on the detection of the perceptual demands of consumers from the target product category. A pan-ethnic-group products form gene clustering method based on emotional semantic is constructed. Consumers' perceptual images of the pan-ethnic-group products are obtained by means of product form gene extraction and coding and computer aided product form clustering technology. A case of form gene clustering about the typical pan-ethnic-group products is investigated which indicates that the method is feasible. This paper opens up a new direction for the future development of product form design which improves the agility of product design process in the era of Industry 4.0.

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

PubMed

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 %).

Genome-Wide Discovery of Genes Required for Capsule Production by Uropathogenic Escherichia coli.

PubMed

Goh, Kelvin G K; Phan, Minh-Duy; Forde, Brian M; Chong, Teik Min; Yin, Wai-Fong; Chan, Kok-Gan; Ulett, Glen C; Sweet, Matthew J; Beatson, Scott A; Schembri, Mark A

2017-10-24

Uropathogenic Escherichia coli (UPEC) is a major cause of urinary tract and bloodstream infections and possesses an array of virulence factors for colonization, survival, and persistence. One such factor is the polysaccharide K capsule. Among the different K capsule types, the K1 serotype is strongly associated with UPEC infection. In this study, we completely sequenced the K1 UPEC urosepsis strain PA45B and employed a novel combination of a lytic K1 capsule-specific phage, saturated Tn 5 transposon mutagenesis, and high-throughput transposon-directed insertion site sequencing (TraDIS) to identify the complement of genes required for capsule production. Our analysis identified known genes involved in capsule biosynthesis, as well as two additional regulatory genes ( mprA and lrhA ) that we characterized at the molecular level. Mutation of mprA resulted in protection against K1 phage-mediated killing, a phenotype restored by complementation. We also identified a significantly increased unidirectional Tn 5 insertion frequency upstream of the lrhA gene and showed that strong expression of LrhA induced by a constitutive Pcl promoter led to loss of capsule production. Further analysis revealed loss of MprA or overexpression of LrhA affected the transcription of capsule biosynthesis genes in PA45B and increased sensitivity to killing in whole blood. Similar phenotypes were also observed in UPEC strains UTI89 (K1) and CFT073 (K2), demonstrating that the effects were neither strain nor capsule type specific. Overall, this study defined the genome of a UPEC urosepsis isolate and identified and characterized two new regulatory factors that affect UPEC capsule production. IMPORTANCE Urinary tract infections (UTIs) are among the most common bacterial infections in humans and are primarily caused by uropathogenic Escherichia coli (UPEC). Many UPEC strains express a polysaccharide K capsule that provides protection against host innate immune factors and contributes to survival

Identification of the recA (tif) gene product of Escherichia coli

PubMed Central

Gudas, Lorraine J.; Mount, David W.

1977-01-01

Treatments that inhibit DNA synthesis in recA+lexA+Escherichia coli stimulate synthesis of a 40,000 molecular weight protein species (protein X). The protein X molecules produced by wild-type and mutant E. coli strains have been compared by two-dimensional gel electrophoresis. One recA mutant (DM1415 spr recA1) produced a protein X with a more acidic isoelectric point than protein X from the wild type, demonstrating that protein X is probably the product of the recA gene. Additional mutants carrying the recA-linked tif-1 mutation yielded a protein X that was more basic than the wild-type protein, indicating that the tif-1 mutation also alters the recA protein. Protein X molecules from the above mutants and wild-type E. coli have been shown to yield similar partial products upon limited proteolysis in sodium dodecyl sulfate, indicating they are the same protein species. These and additional studies suggest that (i) the tif-1 mutation alters a site on the recA protein that is sensitive to DNA synthesis inhibition, (ii) synthesis of recA protein is self-regulated, and (iii) synthesis of recA protein is also regulated by the lexA product with lexA-suppressor mutations such as spr resulting in constitutive synthesis of recA protein. Images PMID:341152

Neurobiology of autism gene products: towards pathogenesis and drug targets.

PubMed

Kleijer, Kristel T E; Schmeisser, Michael J; Krueger, Dilja D; Boeckers, Tobias M; Scheiffele, Peter; Bourgeron, Thomas; Brose, Nils; Burbach, J Peter H

2014-03-01

The genetic heterogeneity of autism spectrum disorders (ASDs) is enormous, and the neurobiology of proteins encoded by genes associated with ASD is very diverse. Revealing the mechanisms on which different neurobiological pathways in ASD pathogenesis converge may lead to the identification of drug targets. The main objective is firstly to outline the main molecular networks and neuronal mechanisms in which ASD gene products participate and secondly to answer the question how these converge. Finally, we aim to pinpoint drug targets within these mechanisms. Literature review of the neurobiological properties of ASD gene products with a special focus on the developmental consequences of genetic defects and the possibility to reverse these by genetic or pharmacological interventions. The regulation of activity-dependent protein synthesis appears central in the pathogenesis of ASD. Through sequential consequences for axodendritic function, neuronal disabilities arise expressed as behavioral abnormalities and autistic symptoms in ASD patients. Several known ASD gene products have their effect on this central process by affecting protein synthesis intrinsically, e.g., through enhancing the mammalian target of rapamycin (mTOR) signal transduction pathway or through impairing synaptic function in general. These are interrelated processes and can be targeted by compounds from various directions: inhibition of protein synthesis through Lovastatin, mTOR inhibition using rapamycin, or mGluR-related modulation of synaptic activity. ASD gene products may all feed into a central process of translational control that is important for adequate glutamatergic regulation of dendritic properties. This process can be modulated by available compounds but may also be targeted by yet unexplored routes.

Increasing meat product functionality by the addition of milled flaxseed Linum usitatissimum.

PubMed

Zając, Marzena; Kulawik, Piotr; Tkaczewska, Joanna; Migdał, Władysław; Pustkowiak, Henryk

2017-07-01

Functional meat products are still rare on the market because it is difficult to incorporate new ingredients and obtain both a healthy and acceptable product. Flaxseed is known for its beneficial properties and, in the present study, it was used as an ingredient in the production of homogenised and liver sausages (0%, 5% and 10% flaxseed addition). Homogenised and liver sausages with the addition of 5% flaxseed were given the highest scores by the consumers, although the colour changed with the addition of flaxseed. The spreadability and hardness of the liver sausages increased with the addition of flaxseed, whereas the texture of homogenised sausages did not change. Addition of flaxseed improved the fatty acids profile from a health point of view for both products, as a result of increasing n-3 fatty acids and overall polyunsaturated fatty acids content. Values for thiobarbituric acid reactive substances were higher in products with flaxseed and were observed to increase during storage. The results of the present study indicate that it is possible to obtain products that are acceptable by consumers and, at the same time, are more healthy. A high level of α-linolenic acid in the sausages at a level of addition of 5% allows the product to be labelled with information regarding their high omega-3 fatty acid content. However, those products are more susceptible to oxidation. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brigham, CJ; Speth, DR; Rha, C

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 selectivelymore » 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.« less

78 FR 77384 - DSM Nutritional Products; Filing of Food Additive Petition (Animal Use)

Federal Register 2010, 2011, 2012, 2013, 2014

2013-12-23

.... FDA-2013-F-1540] DSM Nutritional Products; Filing of Food Additive Petition (Animal Use) AGENCY: Food... (FDA) is announcing that DSM Nutritional Products has filed a petition proposing that the food additive...)), notice is given that a food additive petition (FAP 2277) has been filed by DSM Nutritional Products, 45...

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

PubMed

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.

Production of geranylgeraniol on overexpression of a prenyl diphosphate synthase fusion gene in Saccharomyces cerevisiae.

PubMed

Ohto, Chikara; Muramatsu, Masayoshi; Obata, Shusei; Sakuradani, Eiji; Shimizu, Sakayu

2010-07-01

An acyclic diterpene alcohol, (E,E,E)-geranylgeraniol (GGOH), is one of the important compounds used as perfume and pharmacological agents. A deficiency of squalene (SQ) synthase activity allows yeasts to accumulate an acyclic sesquiterpene alcohol, (E,E)-farnesol, in their cells. Since sterols are essential for the growth of yeasts, a deficiency of SQ synthase activity makes the addition of supplemental sterols to the culture media necessary. To develop a GGOH production method not requiring any supplemental sterols, we overexpressed HMG1 encoding hydroxymethylglutaryl-CoA reductase and the genes of two prenyl diphosphate synthases, ERG20 and BTS1, in Saccharomyces cerevisiae. A prototrophic diploid coexpressing HMG1 and the ERG20-BTS1 fusion accumulated GGOH with neither disruption of the SQ synthase gene nor the addition of any supplemental sterols. The GGOH content on the diploid cultivation in a 5-l jar fermenter reached 138.8 mg/l under optimal conditions.

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

PubMed

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.

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

PubMed

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.

[Collaborative study on regulatory science for facilitating clinical development of gene therapy products for genetic diseases].

PubMed

Uchida, Eriko; Igarashi, Yuka; Sato, Yoji

2014-01-01

Gene therapy products are expected as innovative medicinal products for intractable diseases such as life-threatening genetic diseases and cancer. Recently, clinical developments by pharmaceutical companies are accelerated in Europe and the United States, and the first gene therapy product in advanced countries was approved for marketing authorization by the European Commission in 2012. On the other hand, more than 40 clinical studies for gene therapy have been completed or ongoing in Japan, most of them are conducted as clinical researches by academic institutes, and few clinical trials have been conducted for approval of gene therapy products. In order to promote the development of gene therapy products, revision of the current guideline and/or preparation of concept paper to address the evaluation of the quality and safety of gene therapy products are necessary and desired to clearly show what data should be submitted before First-in-Human clinical trials of novel gene therapy products. We started collaborative study with academia and regulatory agency to promote regulatory science toward clinical development of gene therapy products for genetic diseases based on lentivirus and adeno-associated virus vectors; National Center for Child Health and Development (NCCHD), Nippon Medical School and PMDA have been joined in the task force. At first, we are preparing pre-draft of the revision of the current gene therapy guidelines in this project.

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

PubMed

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

PubMed Central

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-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. PMID:23710454

Association of RUNX2 and TNFSF11 genes with production traits in a paternal broiler line.

PubMed

Grupioni, N V; Stafuzza, N B; Carvajal, A B; Ibelli, A M G; Peixoto, J O; Ledur, M C; Munari, D P

2017-03-22

Intense selection for production traits has improved the genetic gain of important economic traits. However, selection for performance and carcass traits has led to the onset of locomotors problems and decreasing bone strength in broilers. Thus, genes associated with bone integrity traits have become candidates for genetic studies in order to reduce the impact of bone disorders in broilers. This study investigated the association of the RUNX2 and TNFSF11 genes with 79 traits related to performance, carcass composition, organs, and bone integrity in a paternal broiler line. Analyses of genetic association between single-nucleotide polymorphisms (SNPs) and traits were carried out using the maximum likelihood procedures for mixed models. Genetic associations (P < 0.05) were found between SNP g.124,883A>G in the RUNX2 gene and chilled femur weight (additive plus dominance deviation effects within sex) and with performance traits (additive within sex and additive effects). The SNP g.14,862T>C in the TNFSF11 gene presented genetic associations (P < 0.05) with additive plus dominance deviation effects within sex for performance traits. Suggestive genetic associations (P < 0.10) were found with abdominal fat and its yield. Selection based on SNPs g.14,862T>C in TNFSF11 and g.124,883A>G in RUNX2 could be used to improve performance and carcass quality traits in the population studied, although SNP g.14,862T>C was not in Hardy-Weinberg equilibrium because it was not undergoing a selection process. Furthermore, it is important to validate these markers in an unrelated population for use in the selection process.

RBM20 Regulates Circular RNA Production From the Titin Gene.

PubMed

Khan, Mohsin A F; Reckman, Yolan J; Aufiero, Simona; van den Hoogenhof, Maarten M G; van der Made, Ingeborg; Beqqali, Abdelaziz; Koolbergen, Dave R; Rasmussen, Torsten B; van der Velden, Jolanda; Creemers, Esther E; Pinto, Yigal M

2016-10-14

RNA-binding motif protein 20 (RBM20) is essential for normal splicing of many cardiac genes, and loss of RBM20 causes dilated cardiomyopathy. Given its role in splicing, we hypothesized an important role for RBM20 in forming circular RNAs (circRNAs), a novel class of noncoding RNA molecules. To establish the role of RBM20 in the formation of circRNAs in the heart. Here, we performed circRNA profiling on ribosomal depleted RNA from human hearts and identified the expression of thousands of circRNAs, with some of them regulated in disease. Interestingly, we identified 80 circRNAs to be expressed from the titin gene, a gene that is known to undergo highly complex alternative splicing. We show that some of these circRNAs are dynamically regulated in dilated cardiomyopathy but not in hypertrophic cardiomyopathy. We generated RBM20-null mice and show that they completely lack these titin circRNAs. In addition, in a cardiac sample from an RBM20 mutation carrier, titin circRNA production was severely altered. Interestingly, the loss of RBM20 caused only a specific subset of titin circRNAs to be lost. These circRNAs originated from the RBM20-regulated I-band region of the titin transcript. We show that RBM20 is crucial for the formation of a subset of circRNAs that originate from the I-band of the titin gene. We propose that RBM20, by excluding specific exons from the pre-mRNA, provides the substrate to form this class of RBM20-dependent circRNAs. © 2016 American Heart Association, Inc.

Use of Staby® technology for development and production of DNA vaccines free of antibiotic resistance gene

PubMed Central

Reschner, Anca; Scohy, Sophie; Vandermeulen, Gaëlle; Daukandt, Marc; Jacques, Céline; Michel, Benjamin; Nauwynck, Hans; Xhonneux, Florence; Préat, Véronique; Vanderplasschen, Alain; Szpirer, Cédric

2013-01-01

The appearance of new viruses and the cost of developing certain vaccines require that new vaccination strategies now have to be developed. DNA vaccination seems to be a particularly promising method. For this application, plasmid DNA is injected into the subject (man or animal). This plasmid DNA encodes an antigen that will be expressed by the cells of the subject. In addition to the antigen, the plasmid also encodes a resistance to an antibiotic, which is used during the construction and production steps of the plasmid. However, regulatory agencies (FDA, USDA and EMA) recommend to avoid the use of antibiotics resistance genes. Delphi Genetics developed the Staby® technology to replace the antibiotic-resistance gene by a selection system that relies on two bacterial genes. These genes are small in size (approximately 200 to 300 bases each) and consequently encode two small proteins. They are naturally present in the genomes of bacteria and on plasmids. The technology is already used successfully for production of recombinant proteins to achieve higher yields and without the need of antibiotics. In the field of DNA vaccines, we have now the first data validating the innocuousness of this Staby® technology for eukaryotic cells and the feasibility of an industrial production of an antibiotic-free DNA vaccine. Moreover, as a proof of concept, mice have been successfully vaccinated with our antibiotic-free DNA vaccine against a deadly disease, pseudorabies (induced by Suid herpesvirus-1). PMID:24051431

Use of Staby(®) technology for development and production of DNA vaccines free of antibiotic resistance gene.

PubMed

Reschner, Anca; Scohy, Sophie; Vandermeulen, Gaëlle; Daukandt, Marc; Jacques, Céline; Michel, Benjamin; Nauwynck, Hans; Xhonneux, Florence; Préat, Véronique; Vanderplasschen, Alain; Szpirer, Cédric

2013-10-01

The appearance of new viruses and the cost of developing certain vaccines require that new vaccination strategies now have to be developed. DNA vaccination seems to be a particularly promising method. For this application, plasmid DNA is injected into the subject (man or animal). This plasmid DNA encodes an antigen that will be expressed by the cells of the subject. In addition to the antigen, the plasmid also encodes a resistance to an antibiotic, which is used during the construction and production steps of the plasmid. However, regulatory agencies (FDA, USDA and EMA) recommend to avoid the use of antibiotics resistance genes. Delphi Genetics developed the Staby(®) technology to replace the antibiotic-resistance gene by a selection system that relies on two bacterial genes. These genes are small in size (approximately 200 to 300 bases each) and consequently encode two small proteins. They are naturally present in the genomes of bacteria and on plasmids. The technology is already used successfully for production of recombinant proteins to achieve higher yields and without the need of antibiotics. In the field of DNA vaccines, we have now the first data validating the innocuousness of this Staby(®) technology for eukaryotic cells and the feasibility of an industrial production of an antibiotic-free DNA vaccine. Moreover, as a proof of concept, mice have been successfully vaccinated with our antibiotic-free DNA vaccine against a deadly disease, pseudorabies (induced by Suid herpesvirus-1).

Antibacterial Activity of Probiotic Lactobacillus plantarum HK01: Effect of Divalent Metal Cations and Food Additives on Production Efficiency of Antibacterial Compounds.

PubMed

Sharafi, Hakimeh; Alidost, Leila; Lababpour, Abdolmajid; Shahbani Zahiri, Hossein; Abbasi, Habib; Vali, Hojatollah; Akbari Noghabi, Kambiz

2013-06-01

One hundred and sixty lactic acid bacteria, isolated from Iranian traditional dairy products, were screened for antibacterial potential. Among them, an isolate showing remarkable antibacterial activity against both Staphylococcus aureus (PTCC 1112) and Escherichia coli (PTCC 1338) was selected based on minimum inhibitory concentration (AU/mL). The morphological and biochemical characteristics of the isolate matched the literature description about genus Lactobacillus. Partial sequencing of 16S rRNA gene and its alignment with other Lactobacillus strains revealed that the isolate was closely related to the Lactobacillus plantarum. The isolate also exhibited the highest similarity (>99 %) to L. plantarum. We thus tentatively classified the bacterial isolate as L. plantarum HK01. The antibacterial active compound from HK01 strain remained stable for 45 min at 121 °C, and it reached a maximum activity at the end of log phase and the early part of stationary phase. The antibacterial activity of the test isolate, its probiotic properties and production efficacy through addition of some divalent metal cations and food additives were studied as well. The study of bile salt hydrolase (BSH) activity as a function of growth revealed that HK01 strain hydrolysing up to 5 % of sodium salt of glycodeoxycholic acid, correlated with the presence of bsh gene in the isolate. HK01 strain showed high resistance to lysozyme, good adaptation to simulated gastric juice and a moderate bile tolerance. Results obtained from simulated gastric juice conditions showed no significant difference occured during the 70 min. HK01 strain was classified as a strain with low hydrophobicity (34.2 %). Addition of trisodium citrate dehydrates as a food-grade chelator of divalent cations restored antibacterial compound production in MRS broth. Antibacterial compounds of L. plantarum HK01 endured treatment with 10 g/L of SDS, Tween 20, Tween 80 and urea. Concerning food additives, the results

Effect of Periodic Water Addition on Citric Acid Production in Solid State Fermentation

NASA Astrophysics Data System (ADS)

Utpat, Shraddha S.; Kinnige, Pallavi T.; Dhamole, Pradip B.

2013-09-01

Water addition is one of the methods used to control the moisture loss in solid state fermentation (SSF). However, none of the studies report the timing of water addition and amount of water to be added in SSF. Therefore, this work was undertaken with an objective to evaluate the performance of periodic water addition on citric acid production in SSF. Experiments were conducted at different moistures (50-80 %) and temperatures (30-40 °C) to simulate the conditions in a fermenter. Citric acid production by Aspergillus niger (ATCC 9029) using sugarcane baggase was chosen as a model system. Based on the moisture profile, citric acid and sugar data, a strategy was designed for periodic addition of water. Water addition at 48, 96, 144 and 192 h enhanced the citric acid production by 62 % whereas water addition at 72, 120, and 168 h increased the citric acid production by just 17 %.

Database of cattle candidate genes and genetic markers for milk production and mastitis

PubMed Central

Ogorevc, J; Kunej, T; Razpet, A; Dovc, P

2009-01-01

A cattle database of candidate genes and genetic markers for milk production and mastitis has been developed to provide an integrated research tool incorporating different types of information supporting a genomic approach to study lactation, udder development and health. The database contains 943 genes and genetic markers involved in mammary gland development and function, representing candidates for further functional studies. The candidate loci were drawn on a genetic map to reveal positional overlaps. For identification of candidate loci, data from seven different research approaches were exploited: (i) gene knockouts or transgenes in mice that result in specific phenotypes associated with mammary gland (143 loci); (ii) cattle QTL for milk production (344) and mastitis related traits (71); (iii) loci with sequence variations that show specific allele-phenotype interactions associated with milk production (24) or mastitis (10) in cattle; (iv) genes with expression profiles associated with milk production (207) or mastitis (107) in cattle or mouse; (v) cattle milk protein genes that exist in different genetic variants (9); (vi) miRNAs expressed in bovine mammary gland (32) and (vii) epigenetically regulated cattle genes associated with mammary gland function (1). Fourty-four genes found by multiple independent analyses were suggested as the most promising candidates and were further in silico analysed for expression levels in lactating mammary gland, genetic variability and top biological functions in functional networks. A miRNA target search for mammary gland expressed miRNAs identified 359 putative binding sites in 3′UTRs of candidate genes. PMID:19508288

Gene: a gene-centered information resource at NCBI.

PubMed

Brown, Garth R; Hem, Vichet; Katz, Kenneth S; Ovetsky, Michael; Wallin, Craig; Ermolaeva, Olga; Tolstoy, Igor; Tatusova, Tatiana; Pruitt, Kim D; Maglott, Donna R; Murphy, Terence D

2015-01-01

The National Center for Biotechnology Information's (NCBI) Gene database (www.ncbi.nlm.nih.gov/gene) integrates gene-specific information from multiple data sources. NCBI Reference Sequence (RefSeq) genomes for viruses, prokaryotes and eukaryotes are the primary foundation for Gene records in that they form the critical association between sequence and a tracked gene upon which additional functional and descriptive content is anchored. Additional content is integrated based on the genomic location and RefSeq transcript and protein sequence data. The content of a Gene record represents the integration of curation and automated processing from RefSeq, collaborating model organism databases, consortia such as Gene Ontology, and other databases within NCBI. Records in Gene are assigned unique, tracked integers as identifiers. The content (citations, nomenclature, genomic location, gene products and their attributes, phenotypes, sequences, interactions, variation details, maps, expression, homologs, protein domains and external databases) is available via interactive browsing through NCBI's Entrez system, via NCBI's Entrez programming utilities (E-Utilities and Entrez Direct) and for bulk transfer by FTP. Published by Oxford University Press on behalf of Nucleic Acids Research 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.

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

Federal Register 2010, 2011, 2012, 2013, 2014

2011-02-16

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

Ectopic Expression of CDF3 Genes in Tomato Enhances Biomass Production and Yield under Salinity Stress Conditions

PubMed Central

Renau-Morata, Begoña; Molina, Rosa V.; Carrillo, Laura; Cebolla-Cornejo, Jaime; Sánchez-Perales, Manuel; Pollmann, Stephan; Domínguez-Figueroa, José; Corrales, Alba R.; Flexas, Jaume; Vicente-Carbajosa, Jesús; Medina, Joaquín; Nebauer, Sergio G.

2017-01-01

Cycling Dof Factor (CDF) transcription factors (TFs) are involved in multiple processes related to plant growth and development. A member of this family, CDF3, has recently been linked in Arabidopsis to the regulation of primary metabolism and abiotic stress responses, but its role in crop production under stress is still unknown. In this study, we characterized tomato plants overexpressing the CDF3 genes from Arabidopsis and tomato and analyzed their effects on growth and yield under salinity, additionally gaining deeper insights into the molecular function of these TFs. Our results provide evidence for higher biomass production and yield in the 35S::AtCDF3 and 35S::SlCDF3 plants, likely due to a higher photosynthetic capacity resulting in increased sucrose availability. Transcriptome analysis revealed that CDF3 genes regulate a set of genes involved in redox homeostasis, photosynthesis performance and primary metabolism that lead to enhanced biomass production. Consistently, metabolomic profiling revealed that CDF3 evokes changes in the primary metabolism triggering enhanced nitrogen assimilation, and disclosed that the amount of some protective metabolites including sucrose, GABA and asparagine were higher in vegetative tissues of CDF3 overexpressing plants. Altogether these changes improved performance of 35S::AtCDF3 and 35S::SlCDF3 plants under salinity conditions. Moreover, the overexpression of CDF3 genes modified organic acid and sugar content in fruits, improving variables related to flavor perception and fruit quality. Overall, our results associate the CDF3 TF with a role in the control of growth and C/N metabolism, and highlight that overexpression of CDF3 genes can substantially improve plant yield. PMID:28515731

A test strategy for the assessment of additive attributed toxicity of tobacco products.

PubMed

Kienhuis, Anne S; Staal, Yvonne C M; Soeteman-Hernández, Lya G; van de Nobelen, Suzanne; Talhout, Reinskje

2016-08-01

The new EU Tobacco Product Directive (TPD) prohibits tobacco products containing additives that are toxic in unburnt form or that increase overall toxicity of the product. This paper proposes a strategy to assess additive attributed toxicity in the context of the TPD. Literature was searched on toxicity testing strategies for regulatory purposes from tobacco industry and governmental institutes. Although mainly traditional in vivo testing strategies have been applied to assess toxicity of unburnt additives and increases in overall toxicity of tobacco products due to additives, in vitro tests combined with toxicogenomics and validated using biomarkers of exposure and disease are most promising in this respect. As such, tests are needed that are sensitive enough to assess additive attributed toxicity above the overall toxicity of tobacco products, which can associate assay outcomes to human risk and exposure. In conclusion, new, sensitive in vitro assays are needed to conclude whether comparable testing allows for assessment of small changes in overall toxicity attributed to additives. A more pragmatic approach for implementation on a short-term is mandated lowering of toxic emission components. Combined with risk assessment, this approach allows assessment of effectiveness of harm reduction strategies, including banning or reducing of additives. Copyright © 2016 Elsevier Ltd. All rights reserved.

The GermOnline cross-species systems browser provides comprehensive information on genes and gene products relevant for sexual reproduction.

PubMed

Gattiker, Alexandre; Niederhauser-Wiederkehr, Christa; Moore, James; Hermida, Leandro; Primig, Michael

2007-01-01

We report a novel release of the GermOnline knowledgebase covering genes relevant for the cell cycle, gametogenesis and fertility. GermOnline was extended into a cross-species systems browser including information on DNA sequence annotation, gene expression and the function of gene products. The database covers eight model organisms and Homo sapiens, for which complete genome annotation data are available. The database is now built around a sophisticated genome browser (Ensembl), our own microarray information management and annotation system (MIMAS) used to extensively describe experimental data obtained with high-density oligonucleotide microarrays (GeneChips) and a comprehensive system for online editing of database entries (MediaWiki). The RNA data include results from classical microarrays as well as tiling arrays that yield information on RNA expression levels, transcript start sites and lengths as well as exon composition. Members of the research community are solicited to help GermOnline curators keep database entries on genes and gene products complete and accurate. The database is accessible at http://www.germonline.org/.

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

PubMed

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.

Simvastatin and Dipentyl Phthalate Lower Ex vivo Testicular Testosterone Production and Exhibit Additive Effects on Testicular Testosterone and Gene Expression Via Distinct Mechanistic Pathways in the Fetal Rat

EPA Science Inventory

Sex differentiation of the male reproductive tract in mammals is driven, in part, by fetal androgen production. In utero, some phthalate esters (PEs) alter fetal Leydig cell differentiation, reducing the expression of several genes associated with steroid synthesis/transport, and...

78 FR 77384 - DSM Nutritional Products; Filing of Food Additive Petition (Animal Use)

Federal Register 2010, 2011, 2012, 2013, 2014

2013-12-23

.... FDA-2013-F-1539] DSM Nutritional Products; Filing of Food Additive Petition (Animal Use) AGENCY: Food... (FDA) is announcing that DSM Nutritional Products has filed a petition proposing that the food additive... U.S.C. 348(b)(5)), notice is given that a food additive petition (FAP 2276) has been filed by DSM...

77 FR 71750 - DSM Nutritional Products; Filing of Food Additive Petition (Animal Use)

Federal Register 2010, 2011, 2012, 2013, 2014

2012-12-04

.... FDA-2012-F-1100] DSM Nutritional Products; Filing of Food Additive Petition (Animal Use) AGENCY: Food... (FDA) is announcing that DSM Nutritional Products has filed a petition proposing that the food additive...) (21 U.S.C. 348(b)(5))), notice is given that a food additive petition (FAP 2273) has been filed by DSM...

17 CFR 41.25 - Additional conditions for trading for security futures products.

Code of Federal Regulations, 2011 CFR

2011-04-01

... trading for security futures products. 41.25 Section 41.25 Commodity and Securities Exchanges COMMODITY FUTURES TRADING COMMISSION SECURITY FUTURES PRODUCTS Requirements and Standards for Listing Security Futures Products § 41.25 Additional conditions for trading for security futures products. (a) Common...

17 CFR 41.25 - Additional conditions for trading for security futures products.

Code of Federal Regulations, 2012 CFR

2012-04-01

... trading for security futures products. 41.25 Section 41.25 Commodity and Securities Exchanges COMMODITY FUTURES TRADING COMMISSION SECURITY FUTURES PRODUCTS Requirements and Standards for Listing Security Futures Products § 41.25 Additional conditions for trading for security futures products. (a) Common...

17 CFR 41.25 - Additional conditions for trading for security futures products.

Code of Federal Regulations, 2013 CFR

2013-04-01

... trading for security futures products. 41.25 Section 41.25 Commodity and Securities Exchanges COMMODITY FUTURES TRADING COMMISSION SECURITY FUTURES PRODUCTS Requirements and Standards for Listing Security Futures Products § 41.25 Additional conditions for trading for security futures products. (a) Common...

17 CFR 41.25 - Additional conditions for trading for security futures products.

Code of Federal Regulations, 2014 CFR

2014-04-01

... trading for security futures products. 41.25 Section 41.25 Commodity and Securities Exchanges COMMODITY FUTURES TRADING COMMISSION (CONTINUED) SECURITY FUTURES PRODUCTS Requirements and Standards for Listing Security Futures Products § 41.25 Additional conditions for trading for security futures products. (a...

Additive loss-of-function proteasome subunit mutations in CANDLE/PRAAS patients promote type I IFN production

PubMed Central

Brehm, Anja; Liu, Yin; Sheikh, Afzal; Marrero, Bernadette; Omoyinmi, Ebun; Zhou, Qing; Montealegre, Gina; Biancotto, Angelique; Reinhardt, Adam; Almeida de Jesus, Adriana; Pelletier, Martin; Tsai, Wanxia L.; Remmers, Elaine F.; Kardava, Lela; Hill, Suvimol; Kim, Hanna; Lachmann, Helen J.; Megarbane, Andre; Chae, Jae Jin; Brady, Jilian; Castillo, Rhina D.; Brown, Diane; Casano, Angel Vera; Gao, Ling; Chapelle, Dawn; Huang, Yan; Stone, Deborah; Chen, Yongqing; Sotzny, Franziska; Lee, Chyi-Chia Richard; Kastner, Daniel L.; Torrelo, Antonio; Zlotogorski, Abraham; Moir, Susan; Gadina, Massimo; McCoy, Phil; Wesley, Robert; Rother, Kristina; Hildebrand, Peter W.; Brogan, Paul; Krüger, Elke; Aksentijevich, Ivona; Goldbach-Mansky, Raphaela

2015-01-01

Autosomal recessive mutations in proteasome subunit β 8 (PSMB8), which encodes the inducible proteasome subunit β5i, cause the immune-dysregulatory disease chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE), which is classified as a proteasome-associated autoinflammatory syndrome (PRAAS). Here, we identified 8 mutations in 4 proteasome genes, PSMA3 (encodes α7), PSMB4 (encodes β7), PSMB9 (encodes β1i), and proteasome maturation protein (POMP), that have not been previously associated with disease and 1 mutation in PSMB8 that has not been previously reported. One patient was compound heterozygous for PSMB4 mutations, 6 patients from 4 families were heterozygous for a missense mutation in 1 inducible proteasome subunit and a mutation in a constitutive proteasome subunit, and 1 patient was heterozygous for a POMP mutation, thus establishing a digenic and autosomal dominant inheritance pattern of PRAAS. Function evaluation revealed that these mutations variably affect transcription, protein expression, protein folding, proteasome assembly, and, ultimately, proteasome activity. Moreover, defects in proteasome formation and function were recapitulated by siRNA-mediated knockdown of the respective subunits in primary fibroblasts from healthy individuals. Patient-isolated hematopoietic and nonhematopoietic cells exhibited a strong IFN gene-expression signature, irrespective of genotype. Additionally, chemical proteasome inhibition or progressive depletion of proteasome subunit gene transcription with siRNA induced transcription of type I IFN genes in healthy control cells. Our results provide further insight into CANDLE genetics and link global proteasome dysfunction to increased type I IFN production. PMID:26524591

Cloning of the Alcaligenes latus polyhydroxyalkanoate biosynthesis genes and use of these genes for enhanced production of Poly(3-hydroxybutyrate) in Escherichia coli.

PubMed

Choi, J I; Lee, S Y; Han, K

1998-12-01

Polyhydroxyalkanoates (PHAs) are microbial polyesters that can be used as completely biodegradable polymers, but the high production cost prevents their use in a wide range of applications. Recombinant Escherichia coli strains harboring the Ralstonia eutropha PHA biosynthesis genes have been reported to have several advantages as PHA producers compared with wild-type PHA-producing bacteria. However, the PHA productivity (amount of PHA produced per unit volume per unit time) obtained with these recombinant E. coli strains has been lower than that obtained with the wild-type bacterium Alcaligenes latus. To endow the potentially superior PHA biosynthetic machinery to E. coli, we cloned the PHA biosynthesis genes from A. latus. The three PHA biosynthesis genes formed an operon with the order PHA synthase, beta-ketothiolase, and reductase genes and were constitutively expressed from the natural promoter in E. coli. Recombinant E. coli strains harboring the A. latus PHA biosynthesis genes accumulated poly(3-hydroxybutyrate) (PHB), a model PHA product, more efficiently than those harboring the R. eutropha genes. With a pH-stat fed-batch culture of recombinant E. coli harboring a stable plasmid containing the A. latus PHA biosynthesis genes, final cell and PHB concentrations of 194.1 and 141.6 g/liter, respectively, were obtained, resulting in a high productivity of 4.63 g of PHB/liter/h. This improvement should allow recombinant E. coli to be used for the production of PHB with a high level of economic competitiveness.

Structure-Function Analysis of a Broad Specificity Populus trichocarpa Endo-β-glucanase Reveals an Evolutionary Link between Bacterial Licheninases and Plant XTH Gene Products*

PubMed Central

Eklöf, Jens M.; Shojania, Shaheen; Okon, Mark; McIntosh, Lawrence P.; Brumer, Harry

2013-01-01

The large xyloglucan endotransglycosylase/hydrolase (XTH) gene family continues to be the focus of much attention in studies of plant cell wall morphogenesis due to the unique catalytic functions of the enzymes it encodes. The XTH gene products compose a subfamily of glycoside hydrolase family 16 (GH16), which also comprises a broad range of microbial endoglucanases and endogalactanases, as well as yeast cell wall chitin/β-glucan transglycosylases. Previous whole-family phylogenetic analyses have suggested that the closest relatives to the XTH gene products are the bacterial licheninases (EC 3.2.1.73), which specifically hydrolyze linear mixed linkage β(1→3)/β(1→4)-glucans. In addition to their specificity for the highly branched xyloglucan polysaccharide, XTH gene products are distinguished from the licheninases and other GH16 enzyme subfamilies by significant active site loop alterations and a large C-terminal extension. Given these differences, the molecular evolution of the XTH gene products in GH16 has remained enigmatic. Here, we present the biochemical and structural analysis of a unique, mixed function endoglucanase from black cottonwood (Populus trichocarpa), which reveals a small, newly recognized subfamily of GH16 members intermediate between the bacterial licheninases and plant XTH gene products. We postulate that this clade comprises an important link in the evolution of the large plant XTH gene families from a putative microbial ancestor. As such, this analysis provides new insights into the diversification of GH16 and further unites the apparently disparate members of this important family of proteins. PMID:23572521

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hess, Becky M.; Xue, Junfeng; Markillie, Lye Meng

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. Wemore » 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.« less

DHA Production in Escherichia coli by Expressing Reconstituted Key Genes of Polyketide Synthase Pathway from Marine Bacteria.

PubMed

Peng, Yun-Feng; Chen, Wen-Chao; Xiao, Kang; Xu, Lin; Wang, Lian; Wan, Xia

2016-01-01

The gene encoding phosphopantetheinyl transferase (PPTase), pfaE, a component of the polyketide synthase (PKS) pathway, is crucial for the production of docosahexaenoic acid (DHA, 22:6ω3), along with the other pfa cluster members pfaA, pfaB, pfaC and pfaD. DHA was produced in Escherichia coli by co-expressing pfaABCD from DHA-producing Colwellia psychrerythraea 34H with one of four pfaE genes from bacteria producing arachidonic acid (ARA, 20:4ω6), eicosapentaenoic acid (EPA, 20:5ω3) or DHA, respectively. Substitution of the pfaE gene from different strain source in E. coli did not influence the function of the PKS pathway producing DHA, although they led to different DHA yields and fatty acid profiles. This result suggested that the pfaE gene could be switchable between these strains for the production of DHA. The DHA production by expressing the reconstituted PKS pathway was also investigated in different E. coli strains, at different temperatures, or with the treatment of cerulenin. The highest DHA production, 2.2 mg of DHA per gram of dry cell weight or 4.1% of total fatty acids, was obtained by co-expressing pfaE(EPA) from the EPA-producing strain Shewanella baltica with pfaABCD in DH5α. Incubation at low temperature (10-15°C) resulted in higher accumulation of DHA compared to higher temperatures. The addition of cerulenin to the medium increased the proportion of DHA and saturated fatty acids, including C12:0, C14:0 and C16:0, at the expense of monounsaturated fatty acids, including C16:1 and C18:1. Supplementation with 1 mg/L cerulenin resulted in the highest DHA yield of 2.4 mg/L upon co-expression of pfaE(DHA) from C. psychrerythraea.

Coregulation of Terpenoid Pathway Genes and Prediction of Isoprene Production in Bacillus subtilis Using Transcriptomics.

PubMed

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

2013-01-01

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 system level regulation and control of the pathway. To address these limitations, we examined Bacillus subtilis grown under multiple conditions and determined the relationship between altered isoprene production and gene expression patterns. We found that with respect to the amount of isoprene produced, terpenoid genes fall into two distinct subsets with opposing correlations. The group whose expression levels positively correlated with isoprene production included dxs, which is responsible for the commitment step in the pathway, 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. These analyses 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 that accurately predicts production of this secondary metabolite across many simulated environmental conditions.

Coregulation of Terpenoid Pathway Genes and Prediction of Isoprene Production in Bacillus subtilis Using Transcriptomics

PubMed Central

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

2013-01-01

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 system level regulation and control of the pathway. To address these limitations, we examined Bacillus subtilis grown under multiple conditions and determined the relationship between altered isoprene production and gene expression patterns. We found that with respect to the amount of isoprene produced, terpenoid genes fall into two distinct subsets with opposing correlations. The group whose expression levels positively correlated with isoprene production included dxs, which is responsible for the commitment step in the pathway, 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. These analyses 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 that accurately predicts production of this secondary metabolite across many simulated environmental conditions. PMID:23840410

Identification of additive, dominant, and epistatic variation conferred by key genes in cellulose biosynthesis pathway in Populus tomentosa†

PubMed Central

Du, Qingzhang; Tian, Jiaxing; Yang, Xiaohui; Pan, Wei; Xu, Baohua; Li, Bailian; Ingvarsson, Pär K.; Zhang, Deqiang

2015-01-01

Economically important traits in many species generally show polygenic, quantitative inheritance. The components of genetic variation (additive, dominant and epistatic effects) of these traits conferred by multiple genes in shared biological pathways remain to be defined. Here, we investigated 11 full-length genes in cellulose biosynthesis, on 10 growth and wood-property traits, within a population of 460 unrelated Populus tomentosa individuals, via multi-gene association. To validate positive associations, we conducted single-marker analysis in a linkage population of 1,200 individuals. We identified 118, 121, and 43 associations (P< 0.01) corresponding to additive, dominant, and epistatic effects, respectively, with low to moderate proportions of phenotypic variance (R2). Epistatic interaction models uncovered a combination of three non-synonymous sites from three unique genes, representing a significant epistasis for diameter at breast height and stem volume. Single-marker analysis validated 61 associations (false discovery rate, Q ≤ 0.10), representing 38 SNPs from nine genes, and its average effect (R2 = 3.8%) nearly 2-fold higher than that identified with multi-gene association, suggesting that multi-gene association can capture smaller individual variants. Moreover, a structural gene–gene network based on tissue-specific transcript abundances provides a better understanding of the multi-gene pathway affecting tree growth and lignocellulose biosynthesis. Our study highlights the importance of pathway-based multiple gene associations to uncover the nature of genetic variance for quantitative traits and may drive novel progress in molecular breeding. PMID:25428896

Clustered Genes Involved in Cyclopiazonic Acid Production are Next to the Aflatoxin Biosynthesis Gene Cluster in Aspergillus flavus

USDA-ARS?s Scientific Manuscript database

Cyclopiazonic acid (CPA), an indole-tetramic acid toxin, is produced by many species of Aspergillus and Penicillium. In addition to CPA Aspergillus flavus produces polyketide-derived carcinogenic aflatoxins (AFs). AF biosynthesis genes form a gene cluster in a subtelomeric region. Isolates of A. fla...

Enhanced photo-fermentative H2 production using Rhodobacter sphaeroides by ethanol addition and analysis of soluble microbial products

PubMed Central

2014-01-01

Background Biological fermentation routes can provide an environmentally friendly way of producing H2 since they use renewable biomass as feedstock and proceed under ambient temperature and pressure. In particular, photo-fermentation has superior properties in terms of achieving high H2 yield through complete degradation of substrates. However, long-term H2 production data with stable performance is limited, and this data is essential for practical applications. In the present work, continuous photo-fermentative H2 production from lactate was attempted using the purple non-sulfur bacterium, Rhodobacter sphaeroides KD131. As a gradual drop in H2 production was observed, we attempted to add ethanol (0.2% v/v) to the medium. Results As continuous operation went on, H2 production was not sustained and showed a negligible H2 yield (< 0.5 mol H2/mol lactateadded) within two weeks. Electron balance analysis showed that the reason for the gradual drop in H2 production was ascribed to the increase in production of soluble microbial products (SMPs). To see the possible effect of ethanol addition, a batch test was first conducted. The presence of ethanol significantly increased the H2 yield from 1.15 to 2.20 mol H2/mol lactateadded, by suppressing the production of SMPs. The analysis of SMPs by size exclusion chromatography showed that, in the later period of fermentation, more than half of the low molecular weight SMPs (< 1 kDa) were consumed and used for H2 production when ethanol had been added, while the concentration of SMPs continuously increased in the absence of ethanol. It was found that the addition of ethanol facilitated the utilization of reducing power, resulting in an increase in the cellular levels of NAD+ and NADP+. In continuous operation, ethanol addition was effective, such that stable H2 production was attained with an H2 yield of 2.5 mol H2/mol lactateadded. Less than 15% of substrate electrons were used for SMP production, whereas 35% were used in

Expression of Clostridium acetobutylicum ATCC 824 Genes in Escherichia coli for Acetone Production and Acetate Detoxification

PubMed Central

Bermejo, Lourdes L.; Welker, Neil E.; Papoutsakis, Eleftherios T.

1998-01-01

A synthetic acetone operon (ace4) composed of four Clostridium acetobutylicum ATCC 824 genes (adc, ctfAB, and thl, coding for the acetoacetate decarboxylase, coenzyme A transferase, and thiolase, respectively) under the control of the thl promoter was constructed and was introduced into Escherichia coli on vector pACT. Acetone production demonstrated that ace4 is expressed in E. coli and resulted in the reduction of acetic acid levels in the fermentation broth. Since different E. coli strains vary significantly in their growth characteristics and acetate metabolism, ace4 was expressed in three E. coli strains: ER2275, ATCC 11303, and MC1060. Shake flask cultures of MC1060(pACT) produced ca. 2 mM acetone, while both strains ER2275(pACT) and ATCC 11303(pACT) produced ca. 40 mM acetone. Glucose-fed cultures of strain ATCC 11303(pACT) resulted in a 150% increase in acetone titers compared to those of batch shake flask cultures. External addition of sodium acetate to glucose-fed cultures of ATCC 11303(pACT) resulted in further increased acetone titers. In bioreactor studies, acidic conditions (pH 5.5 versus 6.5) improved acetone production. Despite the substantial acetone evaporation due to aeration and agitation in the bioreactor, 125 to 154 mM acetone accumulated in ATCC 11303(pACT) fermentations. These acetone titers are equal to or higher than those produced by wild-type C. acetobutylicum. This is the first study to demonstrate the ability to use clostridial genes in nonclostridial hosts for solvent production. In addition, acetone-producing E. coli strains may be useful hosts for recombinant protein production in that detrimental acetate accumulation can be avoided. PMID:9501448

Genetic improvement of Escherichia coli for ethanol production: Chromosomal integration of Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase II

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ohta, Kazuyoshi; Beall, D.S.; Mejia, J.P.

1991-04-01

Zymomonas mobilis genes for pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhB) were integrated into the Escherichia coli chromosome within or near the pyruvate formate-lyase gene (pfl). Integration improved the stability of the Z. mobilis genes in E. coli, but further selection was required to increase expression. Spontaneous mutants were selected for resistance to high levels of chloramphenicol that also expressed high levels of the Z. mobilis genes. Analogous mutants were selected for increased expression of alcohol dehydrogenase on aldehyde indicator plates. These mutants were functionally equivalent to the previous plasmid-based strains for the fermentation of xylose and glucose tomore » ethanol. Ethanol concentrations of 54.4 and 41.6 g/liter were obtained from 10% glucose and 8% xylose, respectively. The efficiency of conversion exceeded theoretical limits (0.51 g of ethanol/g of sugar) on the basis of added sugars because of the additional production of ethanol from the catabolism of complex nutrients. Further mutations were introduced to inactivate succinate production (frd) and to block homologous recombination (recA).« less

Further improvement in ganoderic acid production in static liquid culture of Ganoderma lucidum by integrating nitrogen limitation and calcium ion addition.

PubMed

Li, Huan-Jun; Zhang, De-Huai; Han, Li-Liang; Yu, Xuya; Zhao, Peng; Li, Tao; Zhong, Jian-Jiang; Xu, Jun-Wei

2016-01-01

To further improve the ganoderic acid (GA) production, a novel integrated strategy by combining nitrogen limitation and calcium ion addition was developed. The effects of the integrated combination on the content of GA-T (one powerful anticancer compound), their intermediates (squalene and lanosterol) and on the transcription levels of GA biosynthetic genes in G. lucidum fermentation were investigated. The maximum GA-T content with the integrated strategy were 1.87 mg/ 100 mg dry cell weight, which was 2.1-4.2 fold higher than that obtained with either calcium ion addition or nitrogen limitation alone, and it is also the highest record as ever reported in submerged fermentation of G. lucidum. The squalene content was increased by 3.9- and 2.2-fold in this case compared with either individual strategy alone. Moreover, the transcription levels of the GA biosynthetic genes encoding 3-hydroxy-3-methyglutaryl coenzyme A reductase and lanosterol synthase were also up-regulated by 3.3-7.5 and 1.3-2.3 fold, respectively.

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

PubMed

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.

Additive Manufacturing and Casting Technology Comparison: Mechanical Properties, Productivity and Cost Benchmark

NASA Astrophysics Data System (ADS)

Vevers, A.; Kromanis, A.; Gerins, E.; Ozolins, J.

2018-04-01

The casting technology is one of the oldest production technologies in the world but in the recent years metal additive manufacturing also known as metal 3D printing has been evolving with huge steps. Both technologies have capabilities to produce parts with internal holes and at first glance surface roughness is similar for both technologies, which means that for precise dimensions parts have to be machined in places where precise fit is necessary. Benchmark tests have been made to find out if parts which are produced with metal additive manufacturing can be used to replace parts which are produced with casting technology. Most of the comparative tests have been made with GJS-400-15 grade which is one of the most popular cast iron grades. To compare mechanical properties samples have been produced using additive manufacturing and tested for tensile strength, hardness, surface roughness and microstructure and then the results have been compared with the samples produced with casting technology. In addition, both technologies have been compared in terms of the production time and production costs to see if additive manufacturing is competitive with the casting technology. The original paper has been written in the Latvian language as part of the Master Thesis within the framework of the production technology study programme at Riga Technical University.

Expression of a DNA Replication Gene Cluster in Bacteriophage T4: Genetic Linkage and the Control of Gene Product Interactions

PubMed Central

Gerald, W. L.; Karam, J. D.

1984-01-01

The results of this study bear on the relationship between genetic linkage and control of interactions between the protein products of different cistrons. In T4 bacteriophage, genes 45 and 44 encode essential components of the phage DNA replication multiprotein complex. T4 gene 45 maps directly upstream of gene 44 relative to the overall direction of reading of this region of the phage chromosome, but it is not known whether these two genes are cotranscribed. It has been shown that a nonsense lesion of T4 gene 45 exerts a cis-dominant inhibitory effect on growth of a missense mutant of gene 44 but not on growth of phage carrying the wild-type gene 44 allele. In previous work, we confirmed these observations on polarity of the gene 45 mutation but detected no polar effects by this lesion on synthesis of either mutant or wild-type gene 44 protein. In the present study, we demonstrate that mRNA for gene 44 protein is separable by gel electrophoresis from gene 45-protein-encoding mRNA. That is, the two proteins are not synthesized from one polycistronic message, and the cis-dominant inhibitory effect of the gene 45 mutation on gene 44 function is probably expressed at a posttranslational stage. We propose that close genetic linkage, whether or not it provides shared transcriptional and translational regulatory signals for certain clusters of functionally related cistrons, may determine the intracellular compartmentalization for synthesis of proteins encoded by these clusters. In prokaryotes, such linkage-dependent compartmentation may minimize the diffusion distances between gene products that are synthesized at low levels and are destined to interact. PMID:6745641

The role of histamine degradation gene polymorphisms in moderating the effects of food additives on children's ADHD symptoms.

PubMed

Stevenson, Jim; Sonuga-Barke, Edmund; McCann, Donna; Grimshaw, Kate; Parker, Karen M; Rose-Zerilli, Matthew J; Holloway, John W; Warner, John O

2010-09-01

Food additives can exacerbate ADHD symptoms and cause non-immunoglobulin E-dependent histamine release from circulating basophils. However, children vary in the extent to which their ADHD symptoms are exacerbated by the ingestion of food additives. The authors hypothesized that genetic polymorphisms affecting histamine degradation would explain the diversity of responses to additives. In a double-blind, placebo-controlled crossover trial, challenges involving two food color additive and sodium benzoate (preservative) mixtures in a fruit drink were administered to a general community sample of 3-year-old children (N = 153) and 8/9-year-old children (N = 144). An aggregate ADHD symptom measure (based on teacher and parent blind ratings of behavior, blind direct observation of behavior in the classroom, and--for 8/9-year-old children only--a computerized measure of attention) was the main outcome variable. The adverse effect of food additives on ADHD symptoms was moderated by histamine degradation gene polymorphisms HNMT T939C and HNMT Thr105Ile in 3- and 8/9-year-old children and by a DAT1 polymorphism (short versus long) in 8/9-year-old children only. There was no evidence that polymorphisms in catecholamine genes COMT Val108Met, ADRA2A C1291G, and DRD4-rs7403703 moderated the effect on ADHD symptoms. Histamine may mediate the effects of food additives on ADHD symptoms, and variations in genes influencing the action of histamine may explain the inconsistency between previous studies. Genes influencing a range of neurotransmitter systems and their interplay with environmental factors, such as diet, need to be examined to understand genetic influences on ADHD symptoms.

Carbon repression of cellobiose dehydrogenase production in the white rot fungus Trametes versicolor is mediated at the level of gene transcription.

PubMed

Stapleton, P C; Dobson, A D W

2003-04-25

Cellobiose dehydrogenase (CDH) production in Trametes versicolor is induced in the presence of cellulose, but decreases when additional carbon sources such as glucose and maltose are added to the fungal cultures. Using T. versicolor-specific cdh primers in a reverse transcription-polymerase chain reaction-based approach, it appears that this repression in CDH production is being mediated at the level of gene transcription. When a 1.6-kb upstream region of the T. versicolor cdh gene was cloned and sequenced, a number of putative CreA-like binding sites were observed. We propose that these sites may be involved in mediating this repressive effect, based on their similarity to the consensus [5'-SYGGRGG-3'] site for binding of the CreA and Cre1 repressor proteins.

Innovations in value-addition of edible meat by-products.

PubMed

Toldrá, Fidel; Aristoy, M-Concepción; Mora, Leticia; Reig, Milagro

2012-11-01

While muscle foods are the more commonly consumed portion of an animal, meat by-products such as the entrails and internal organs are also widely consumed. Considered high-priced delicacies or waste material to be tossed away, the use and value of offal-edible and inedible meat by-products depend entirely on the culture and country in question. The skin, blood, bones, meat trimmings, fatty tissues, horns, hoofs, feet, skull, and internal organs of harvested animals comprise a wide variety of products including human or pet food or processed materials in animal feed, fertilizer, or fuel. Industry is using science and innovation to add value to animal by-products far beyond its usual profitability. Regardless of the final product's destination, it is still necessary to employ the most up-to-date and effective tools to analyze these products for nutritional properties, to search for key active molecules in nutrition like bioactive peptides, food safety (antimicrobial peptides), medicine, cosmetics or other fields, to develop new technological applications and to continue innovation towards advanced value-addition of meat by-products. Copyright © 2012 Elsevier Ltd. All rights reserved.

An Inventory of Methods for the Assessment of Additive Increased Addictiveness of Tobacco Products

PubMed Central

van de Nobelen, Suzanne; Kienhuis, Anne S.

2016-01-01

Background: Cigarettes and other forms of tobacco contain the addictive drug nicotine. Other components, either naturally occurring in tobacco or additives that are intentionally added during the manufacturing process, may add to the addictiveness of tobacco products. As such, these components can make cigarette smokers more easily and heavily dependent. Efforts to regulate tobacco product dependence are emerging globally. Additives that increase tobacco dependence will be prohibited under the new European Tobacco Product Directive. Objective: This article provides guidelines and recommendations for developing a regulatory strategy for assessment of increase in tobacco dependence due to additives. Relevant scientific literature is summarized and criteria and experimental studies that can define increased dependence of tobacco products are described. Conclusions: Natural tobacco smoke is a very complex matrix of components, therefore analysis of the contribution of an additive or a combination of additives to the level of dependence on this product is challenging. We propose to combine different type of studies analyzing overall tobacco product dependence potential and the functioning of additives in relation to nicotine. By using a combination of techniques, changes associated with nicotine dependence such as behavioral, physiological, and neurochemical alterations can be examined to provide sufficient information. Research needs and knowledge gaps will be discussed and recommendations will be made to translate current knowledge into legislation. As such, this article aids in implementation of the Tobacco Product Directive, as well as help enable regulators and researchers worldwide to develop standards to reduce dependence on tobacco products. Implications: This article provides an overall view on how to assess tobacco product constituents for their potential contribution to use and dependence. It provides guidelines that help enable regulators worldwide to

Orsomucoid: A new variant and additional duplicated ORM1 gene in Qatari population

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sebetan, I.M.; Alali, K.A.; Alzaman, A.

1994-09-01

A new genetically determined ORM2 variant and additional duplicated ORM1 gene were observed in Qatari population using isoelectric focusing in ultra thin layer polyacrylamide gels. The studied population samples indicate occurence of six ORM1 alleles and three ORM2 ones. A simple reliable method for separation of orsomucoid variations with comparison of different reported methods will be presented.

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

NASA Astrophysics Data System (ADS)

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.

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

PubMed

Cary, Jeffrey W; Harris-Coward, Pamela; Scharfenstein, Leslie; Mack, Brian M; Chang, Perng-Kuang; Wei, Qijian; Lebar, Matthew; Carter-Wientjes, Carol; Majumdar, Rajtilak; Mitra, Chandrani; Banerjee, Sourav; Chanda, Anindya

2017-10-12

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 Δ hbx1 mutants did not produce conidiophores. The inability of Δ hbx1 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.

Effect of additives on mineral trioxide aggregate setting reaction product formation.

PubMed

Zapf, Angela M; Chedella, Sharath C V; Berzins, David W

2015-01-01

Mineral trioxide aggregate (MTA) sets via hydration of calcium silicates to yield calcium silicate hydrates and calcium hydroxide (Ca[OH]2). However, a drawback of MTA is its long setting time. Therefore, many additives have been suggested to reduce the setting time. The effect those additives have on setting reaction product formation has been ignored. The objective was to examine the effect additives have on MTA's setting time and setting reaction using differential scanning calorimetry (DSC). MTA powder was prepared with distilled water (control), phosphate buffered saline, 5% calcium chloride (CaCl2), 3% sodium hypochlorite (NaOCl), or lidocaine in a 3:1 mixture and placed in crucibles for DSC evaluation. The setting exothermic reactions were evaluated at 37°C for 8 hours to determine the setting time. Separate samples were stored and evaluated using dynamic DSC scans (37°C→640°C at10°C/min) at 1 day, 1 week, 1 month, and 3 months (n = 9/group/time). Dynamic DSC quantifies the reaction product formed from the amount of heat required to decompose it. Thermographic peaks were integrated to determine enthalpy, which was analyzed with analysis of variance/Tukey test (α = 0.05). Isothermal DSC identified 2 main exothermal peaks occurring at 44 ± 12 and 343 ± 57 minutes for the control. Only the CaCl2 additive was an accelerant, which was observed by a greater exothermic peak at 101 ± 11 minutes, indicating a decreased setting time. The dynamic DSC scans produced an endothermic peak around 450°C-550°C attributed to Ca(OH)2 decomposition. The use of a few additives (NaOCl and lidocaine) resulted in significantly less Ca(OH)2 product formation. DSC was used to discriminate calcium hydroxide formation in MTA mixed with various additives and showed NaOCl and lidocaine are detrimental to MTA reaction product formation, whereas CaCl2 accelerated the reaction. Copyright © 2015 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

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

PubMed

Purvis, Ian William; Franklin, Ian Robert

2005-01-01

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.

Older paternal age and fresh gene mutation: data on additional disorders.

PubMed

Jones, K L; Smith, D W; Harvey, M A; Hall, B D; Quan, L

1975-01-01

Older paternal age has previously been documented as a factor in sporadic fresh mutational cases of several autosomal dominant disorders. In this collaborative study, an older mean paternal age has been documented in sporadic cases of at least five additional dominantly inheritable disorders; the basal cell nevus syndrome, the Waardenburg syndrome, the Crouzon syndrome, the oculo-dental-digital sysdrome, and the Treacher-Collins syndrome. It was also found to be a factor in acrodysostosis and progeria, suggesting a fresh mutant gene etiology for these two conditions in which virtually all cases have been sporadic and the mode of genetic etiology has been unknown.

Characterization of monocarboxylate transporter 1 (MCT1) binding affinity for Basigin gene products and L1cam.

PubMed

Howard, John; Finch, Nicole A; Ochrietor, Judith D

2010-07-01

The purpose of this study was to determine the binding affinities of Basigin gene products and neural cell adhesion molecule L1cam for monocarboxylate transporter-1 (MCT1). ELISA binding assays were performed in which recombinant proteins of the transmembrane domains of Basigin gene products and L1cam were incubated with MCT1 captured from mouse brain. It was determined that Basigin gene products bind MCT1 with moderate affinity, but L1cam does not bind MCT1. Despite a high degree of sequence conservation between Basigin gene products and L1cam, the sequences are different enough to prevent L1cam from interacting with MCT1.

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

PubMed

Alvarez, Marco A; Qi, Xiaojun; Yan, Changhui

2011-07-29

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. 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. 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.

Natural Products Version 2.0: Connecting Genes to Molecules

PubMed Central

Walsh, Christopher T.; Fischbach, Michael A.

2009-01-01

Natural products have played a prominent role in the history of organic chemistry, and they continue to be important as drugs, biological probes, and targets of study for synthetic and analytical chemists. In this perspective, we explore how connecting Nature’s small molecules to the genes that encode them has sparked a renaissance in natural product research, focusing primarily on the biosynthesis of polyketides and nonribosomal peptides. We survey monomer biogenesis, coupling chemistries from templated and non-templated pathways, and the broad set of tailoring reactions and hybrid pathways that give rise to the diverse scaffolds and functionalization patterns of natural products. We conclude by considering two questions: What would it take to find all natural product scaffolds? What kind of scientists will be studying natural products in the future? PMID:20121095

Production of indole antibiotics induced by exogenous gene derived from sponge metagenomes.

PubMed

Takeshige, Yuya; Egami, Yoko; Wakimoto, Toshiyuki; Abe, Ikuro

2015-05-01

Sponge metagenomes are accessible genetic sources containing genes and gene clusters responsible for the biosynthesis of sponge-derived bioactive natural products. In this study, we obtained the clone pDC112, producing turbomycin A and 2,2-di(3-indolyl)-3-indolone, based on the functional screening of the metagenome library derived from the marine sponge Discodermia calyx. The subcloning experiment identified ORF 25, which is homologous to inosine 5'-monophosphate dehydrogenase and required for the production of 2,2-di(3-indolyl)-3-indolone in Escherichia coli.

Wogonin but not Nor-wogonin inhibits lipopolysaccharide and lipoteichoic acid-induced iNOS gene expression and NO production in macrophages.

PubMed

Huang, Guan-Cheng; Chow, Jyh-Ming; Shen, Shing-Chuan; Yang, Liang-Yo; Lin, Cheng-Wei; Chen, Yen-Chou

2007-08-01

Wogonin (Wog; 5,7-dihydroxy-8-methoxy flavone) has been shown to effectively inhibit lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) gene expression and nitric oxide production in our previous study. In the present study, we found that Nor-wogonin (N-Wog; 5,7,8-trihydroxyl flavone), a structural analogue of Wog with an OH substitution at C8, performed different effect on LPS- or lipoteichoic acid (LTA)-induced iNOS gene expression and nitric oxide (NO) production in macrophages. Wog, but not N-Wog, significantly inhibits LPS- or LTA-induced NO production through suppressing iNOS gene expression at both protein and mRNA without affecting NO donor sodium nitroprusside-induced NO production, NOS enzyme activity, and cells viability. Activation of JNKs (not ERKs) via phosphorylation induction, and an increase in c-Jun (not c-Fos) protein expression were involved in LPS- and LTA-treated RAW264.7 cells, and those events were blocked by Wog, but not N-Wog, addition. Furthermore, 5,7-diOH flavone, but not 5-OH flavone, 7-OH flavone, 5-OH-7-OCH(3) flavone, significantly inhibits LPS-induced iNOS protein expression and NO production, and 7,8-diOCH(3) flavone performs more effective inhibitory activity on LPS-induced NO production and iNOS protein expression than 7-OCH(3)-8-OH flavone. These data suggest that OHs at both C5 and C7 are essential for NO inhibition of flavonoids, and OCH(3) at C8 may contribute to this activity, and suppression of JNKs-c-Jun activation is involved.

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

PubMed

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.

A homomeric geranyl diphosphate synthase-encoding gene from Camptotheca acuminata and its combinatorial optimization for production of geraniol in Escherichia coli.

PubMed

Yang, Lixia; Jiang, Liangzhen; Li, Wei; Yang, Yun; Zhang, Guolin; Luo, Yinggang

2017-10-01

Geranyl diphosphate (GPP), the unique precursor for all monoterpenoids, is biosynthesized from isopentenyl diphosphate and dimethylallyl diphosphate via the head-to-tail condensation reaction catalyzed by GPP synthase (GPPS). Herein a homomeric GPPS from Camptotheca acuminata, a camptothecin-producing plant, was obtained from 5'- and 3'-rapid amplification of cDNA ends and subsequent overlap extension and convenient PCR amplifications. The truncate CaGPPS was introduced to replace ispA of pBbA5c-MevT(CO)-MBIS(CO, ispA), a de novo biosynthetic construct for farnesyl diphosphate generation, and overexpressed in Escherichia coli, together with the truncate geraniol synthase-encoding gene from C. acuminata (tCaGES), to confirm CaGPPS-catalyzed reaction in vivo. A 24.0 ± 1.3 mg L -1 of geraniol was produced in the recombinant E. coli. The production of GPP was also validated by the direct UPLC-HRMS E analyses. The tCaGPPS and tCaGES genes with different copy numbers were introduced into E. coli to balance their catalytic potential for high-yield geraniol production. A 1.6-fold increase of geraniol production was obtained when four copies of tCaGPPS and one copy of tCaGES were introduced into E. coli. The following fermentation conditions optimization, including removal of organic layers and addition of new n-decane, led to a 74.6 ± 6.5 mg L -1 of geraniol production. The present study suggested that the gene copy number optimization, i.e., the ratio of tCaGPPS and tCaGES, plays an important role in geraniol production in the recombinant E. coli. The removal and addition of organic solvent are very useful for sustainable high-yield production of geraniol in the recombinant E. coli in view of that the solubility of geraniol is limited in the fermentation broth and/or n-decane.

Qualitative identification of permitted and non-permitted colour additives in food products.

PubMed

Harp, Bhakti Petigara; Miranda-Bermudez, Enio; Baron, Carolina I; Richard, Gerald I

2012-01-01

Colour additives are dyes, pigments or other substances that can impart colour when added or applied to food, drugs, cosmetics, medical devices, or the human body. The substances must be pre-approved by the US Food and Drug Administration (USFDA) and listed in Title 21 of the US Code of Federal Regulations before they may be used in products marketed in the United States. Some also are required to be batch certified by the USFDA prior to their use. Both domestic and imported products sold in interstate commerce fall under USFDA jurisdiction, and the USFDA's district laboratories use a combination of analytical methods for identifying or confirming the presence of potentially violative colour additives. We have developed a qualitative method for identifying 17 certifiable, certification exempt, and non-permitted colour additives in various food products. The method involves extracting the colour additives from a product and isolating them from non-coloured components with a C(18) Sep-Pak cartridge. The colour additives are then separated and identified by liquid chromatography (LC) with photodiode array detection, using an Xterra RP18 column and gradient elution with aqueous ammonium acetate and methanol. Limits of detection (LODs) ranged from 0.02 to 1.49 mg/l. This qualititative LC method supplements the visible spectrophotometric and thin-layer chromatography methods currently used by the USFDA's district laboratories and is less time-consuming and requires less solvent compared to the other methods. The extraction step in the new LC method is a simple and an efficient process that can be used for most food types.

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

PubMed

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.

Re-engineering bacteria for ethanol production

DOEpatents

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.

15 CFR 713.4 - Advance declaration requirements for additionally planned production, processing, or consumption...

Code of Federal Regulations, 2010 CFR

2010-01-01

... INDUSTRY AND SECURITY, DEPARTMENT OF COMMERCE CHEMICAL WEAPONS CONVENTION REGULATIONS ACTIVITIES INVOLVING SCHEDULE 2 CHEMICALS § 713.4 Advance declaration requirements for additionally planned production... additionally planned production, processing, or consumption of Schedule 2 chemicals. 713.4 Section 713.4...

15 CFR 713.4 - Advance declaration requirements for additionally planned production, processing, or consumption...

Code of Federal Regulations, 2011 CFR

2011-01-01

... INDUSTRY AND SECURITY, DEPARTMENT OF COMMERCE CHEMICAL WEAPONS CONVENTION REGULATIONS ACTIVITIES INVOLVING SCHEDULE 2 CHEMICALS § 713.4 Advance declaration requirements for additionally planned production... additionally planned production, processing, or consumption of Schedule 2 chemicals. 713.4 Section 713.4...

FlyBase: genes and gene models

PubMed Central

Drysdale, Rachel A.; Crosby, Madeline A.

2005-01-01

FlyBase (http://flybase.org) is the primary repository of genetic and molecular data of the insect family Drosophilidae. For the most extensively studied species, Drosophila melanogaster, a wide range of data are presented in integrated formats. Data types include mutant phenotypes, molecular characterization of mutant alleles and aberrations, cytological maps, wild-type expression patterns, anatomical images, transgenic constructs and insertions, sequence-level gene models and molecular classification of gene product functions. There is a growing body of data for other Drosophila species; this is expected to increase dramatically over the next year, with the completion of draft-quality genomic sequences of an additional 11 Drosphila species. PMID:15608223

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

PubMed

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.

Single gene insertion drives bioalcohol production by a thermophilic archaeon

PubMed Central

Basen, Mirko; Schut, Gerrit J.; Nguyen, Diep M.; Lipscomb, Gina L.; Benn, Robert A.; Prybol, Cameron J.; Vaccaro, Brian J.; Poole, Farris L.; Kelly, Robert M.; Adams, Michael W. W.

2014-01-01

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 °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. PMID:25368184

Single gene insertion drives bioalcohol production by a thermophilic archaeon

DOE Office of Scientific and Technical Information (OSTI.GOV)

Basen, M; Schut, GJ; Nguyen, DM

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. Bymore » 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.« less

Vasopressin Gene-Related Products in the Management of Breast Cancer

DTIC Science & Technology

1997-10-01

information added to an absence of vasopressin gene-related products from fibrocystic disease should provide us with a new screening test for...official Department of Army endorsement or approval of the products or services of these organizations. In conducting research using animals , the...investigator (s) "äclhered to the "Guide for the Care and Use of Laboratory Animals ," prepared by the Committee on Care and use of Laboratory

fabH deletion increases DHA production in Escherichia coli expressing Pfa genes.

PubMed

Giner-Robles, Laura; Lázaro, Beatriz; de la Cruz, Fernando; Moncalián, Gabriel

2018-06-08

Some marine bacteria, such as Moritella marina, produce the nutraceutical docosahexaenoic acid (DHA) thanks to a specific enzymatic complex called Pfa synthase. Escherichia coli heterologously expressing the pfa gene cluster from M. marina also produces DHA. The aim of this study was to find genetic or metabolic conditions to increase DHA production in E. coli. First, we analysed the effect of the antibiotic cerulenin, showing that DHA production increased twofold. Then, we tested a series of single gene knockout mutations affecting fatty acid biosynthesis, in order to optimize the synthesis of DHA. The most effective mutant, fabH, showed a threefold increase compared to wild type strain. The combination of cerulenin inhibition and fabH deletion rendered a 6.5-fold improvement compared to control strain. Both strategies seem to have the same mechanism of action, in which fatty acid synthesis via the canonical pathway (fab pathway) is affected in its first catalytic step, which allows the substrates to be used by the heterologous pathway to synthesize DHA. DHA-producing E. coli strain that carries a fabH gene deletion boosts DHA production by tuning down the competing canonical biosynthesis pathway. Our approach can be used for optimization of DHA production in different organisms.

Market surveillance on non-halal additives incorporated in surimi based products using polymerase chain reaction (PCR)-southern hybridization analysis

NASA Astrophysics Data System (ADS)

Aravindran, S.; Sahilah, A. M.; Aminah, A.

2014-09-01

Halal surveillance on halal ingredients incorporated in surimi based products were studied using polymerase chain reaction (PCR)-southern hybridization on chip analysis. The primers used in this technique were targeted on mitochondria DNA (mtDNA) of cytochrome b (cyt b) gene sequence which able to differentiate 7 type (beef, chicken, duck, goat, buffalo, lamb and pork) of species on a single chip. 17 (n = 17*3) different brands of surimi-based product were purchased randomly from Selangor local market in January 2013. Of 17 brands, 3 (n = 3*3) brands were positive for chicken DNA, 1 (n = 1*3) brand was positive for goat DNA, and the remainder 13 brands (n = 13*3) have no DNA species detected. The sensitivity of PCR-southern hybridization primers to detect each meat species was 0.1 ng. In the present study, it is evidence that PCR-Southern Hybridization analysis offered a reliable result due to its highly specific and sensitive properties in detecting non-halal additive such as plasma protein incorporation in surimi-based product.

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

PubMed

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.

Diversity, Distribution and Quantification of Antibiotic Resistance Genes in Goat and Lamb Slaughterhouse Surfaces and Meat Products

PubMed Central

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. PMID:25479100

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

PubMed

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.

The differentiation of tuna (family: Scombridae) products through the PCR-based analysis of the cytochrome b gene and parvalbumin introns.

PubMed

Abdullah, Asadatun; Rehbein, Hartmut

2016-01-30

In spite of the many studies performed over the years, there are still problems in the authentication of closely related tuna species, not only for canned fish but also for raw products. With the aim of providing screening methods to identify different tuna species and related scombrids, segments of mitochondrial cytochrome b (cyt b) and nuclear parvalbumin genes were amplified and sequenced or subjected to single-strand conformation polymorphism (SSCP) and restriction fragment length polymorphism (RFLP) analyses. The nucleotide diagnostic sites in the cyt b gene of five tuna species from Indonesia were determined in this study and used to construct a phylogenetic tree. In addition, the suitability of the nuclear gene that encodes parvalbumin for the differentiation of tuna species was determined by SSCP and RFLP analyses of an intron segment. RFLP differentiated Thunnus albacares and from T. obesus, and fish species in the Thunnus genus could be distinguished from bullet tuna (Auxis rochei) by SSCP. Parvalbumin-based polymerase chain reaction systems could serve as an additional tool in the detection and identification of tuna and other Scombridae fish species for routine seafood control. This reaction can be performed in addition to the cyt b analysis as previously described. © 2015 Society of Chemical Industry.

Production of recombinant adenovirus containing human interlukin-4 gene.

PubMed

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

2011-11-01

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 that made it a good choice for using in cancer gene therapy, controlling inflammatory diseases, and studies on autoimmune diseases. In brief, IL-4 coding sequence was amplified by and cloned in pAd-Track-CMV. Then, by means of homologous recombination between recombinant pAd-Track-CMV and Adeasy-1 plasmid in bacteria, recombinant adenovirus complete genome was made and IL-4 containing shuttle vector was incorporated into the viral backbone. After linearization, for virus packaging, viral genome was transfected into HEK-293 cell line. Viral production was conveniently followed with the aid of green fluorescent protein. Recombinant adenovirus produced here, was capable to infecting cell lines and express interlukin-4 in cell. This system can be used as a powerful, easy, and cost benefit tool in various studies on cancer gene therapy and also studies on immunogenetics.

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

PubMed Central

Scott, Barry; Young, Carolyn A.; Saikia, Sanjay; McMillan, Lisa K.; Monahan, Brendon J.; Koulman, Albert; Astin, Jonathan; Eaton, Carla J.; Bryant, Andrea; Wrenn, Ruth E.; Finch, Sarah C.; Tapper, Brian A.; Parker, Emily J.; Jameson, Geoffrey B.

2013-01-01

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. PMID:23949005

Analysis of the K1 capsule biosynthesis genes of Escherichia coli: definition of three functional regions for capsule production.

PubMed

Boulnois, G J; Roberts, I S; Hodge, R; Hardy, K R; Jann, K B; Timmis, K N

1987-06-01

Transposon and deletion analysis of the cloned K1 capsule biosynthesis genes of Escherichia coli revealed that approximately 17 kb of DNA, split into three functional regions, is required for capsule production. One block (region 1) is required for translocation of polysaccharide to the cell surface and mutations in this region result in the intracellular appearance of polymer indistinguishable on immunoelectrophoresis to that found on the surface of K1 encapsulated bacteria. This material was released from the cell by osmotic shock indicating that the polysaccharide was probably present in the periplasmic space. Insertions in a second block (region 2) completely abolished polymer production and this second region is believed to encode the enzymes for the biosynthesis and polymerisation of the K1 antigen. Addition of exogenous N-acetylneuraminic acid to one insertion mutant in this region restored its ability to express surface polymer as judged by K1 phage sensitivity. This insertion probably defines genes involved in biosynthesis of N-acetylneuraminic acid. Insertions in a third block (region 3) result in the intracellular appearance of polysaccharide with a very low electrophoretic mobility. The presence of the cloned K1 capsule biosynthesis genes on a multicopy plasmid in an E. coli K-12 strain did not increase the yields of capsular polysaccharide produced compared to the K1+ isolate from which the genes were cloned.

Phytate addition to soil induces changes in the abundance and expression of Bacillus β-propeller phytase genes in the rhizosphere.

PubMed

Jorquera, Milko A; Saavedra, Nicolás; Maruyama, Fumito; Richardson, Alan E; Crowley, David E; del C Catrilaf, Rosa; Henriquez, Evelyn J; de la Luz Mora, María

2013-02-01

Phytate-mineralizing rhizobacteria (PMR) perform an essential function for the mineralization of organic phosphorus but little is known about their ecology in soils and rhizosphere. In this study, PCR-based methods were developed for detection and quantification of the Bacillus β-propeller phytase (BPP) gene. Experiments were conducted to monitor the presence and persistence of a phytate-mineralizing strain, Bacillus sp. MQH19, after inoculation of soil microcosms and within the rhizosphere. The occurrence of the BPP gene in natural pasture soils from Chilean Andisols was also examined. The results showed that the Bacillus BPP gene was readily detected in sterile and nonsterile microcosms, and that the quantitative PCR (qPCR) methods could be used to monitor changes in the abundance of the BPP gene over time. Our results also show that the addition of phytate to nonsterile soils induced the expression of the BPP gene in the rhizosphere of ryegrass and the BPP gene was detected in all pasture soils sampled. This study shows that phytate addition soils induced changes in the abundance and expression of Bacillus BPP to genes in the rhizosphere and demonstrates that Bacillus BPP gene is cosmopolitan in pasture soils from Chilean Andisols. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

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

PubMed

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.

Another face of the Treacher Collins syndrome (TCOF1) gene: identification of additional exons.

PubMed

So, Rolando B; Gonzales, Bianca; Henning, Dale; Dixon, Jill; Dixon, Michael J; Valdez, Benigno C

2004-03-17

Treacher Collins syndrome (TCS) is characterized by an abnormality in craniofacial development during early embryogenesis. TCS is caused by mutations in the gene TCOF1, which encodes the nucleolar phosphoprotein treacle. Genetic and proteomic characterizations of TCS/treacle are based on the previously reported 26 exons of TCOF1. Here, we report the identification of 231-nucleotide (nt) exon 6A (between exons 6 and 7) and 108-nt exon 16A (between exons 16 and 17). Isoforms with exon 6A are up to 3.7-fold more abundant than alternatively spliced variants without exon 6A, but only minor isoforms contain exon 16A. Exon 6A encodes a peptide sequence containing basic and acidic domains similar to 10 other exons of TCOF1. Unlike the other exons, exon 6A encodes a nuclear localization signal (NLS) which does not, however, alter the nucleolar localization of full-length treacle. The discovery of exons 6A and 16A is relevant to mutational analysis of the TCOF1 gene in TCS patients, and to functional analysis of its gene product.

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

PubMed

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.

Metabolic engineering of Escherichia coli for ethanol production without foreign genes

NASA Astrophysics Data System (ADS)

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

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

DOEpatents

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.

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

DOEpatents

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

2011-10-04

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.

Engineering Neurospora crassa for cellobionate production directly from cellulose without any enzyme addition

USDA-ARS?s Scientific Manuscript database

A previously engineered strain of N. crassa F5'ace-1'cre-1'ndvB) with six out of seven ß-glucosidase (bgl) genes, two transcription factors (cre1 and ace-1) and cellobionate phosphorylase (ndvB) deleted was able to produce cellobiose and cellobionate directly from cellulose without the addition of e...

Gene function prediction based on Gene Ontology Hierarchy Preserving Hashing.

PubMed

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.

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

PubMed

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.

DynGO: a tool for visualizing and mining of Gene Ontology and its associations

PubMed Central

Liu, Hongfang; Hu, Zhang-Zhi; Wu, Cathy H

2005-01-01

Background A large volume of data and information about genes and gene products has been stored in various molecular biology databases. A major challenge for knowledge discovery using these databases is to identify related genes and gene products in disparate databases. The development of Gene Ontology (GO) as a common vocabulary for annotation allows integrated queries across multiple databases and identification of semantically related genes and gene products (i.e., genes and gene products that have similar GO annotations). Meanwhile, dozens of tools have been developed for browsing, mining or editing GO terms, their hierarchical relationships, or their "associated" genes and gene products (i.e., genes and gene products annotated with GO terms). Tools that allow users to directly search and inspect relations among all GO terms and their associated genes and gene products from multiple databases are needed. Results We present a standalone package called DynGO, which provides several advanced functionalities in addition to the standard browsing capability of the official GO browsing tool (AmiGO). DynGO allows users to conduct batch retrieval of GO annotations for a list of genes and gene products, and semantic retrieval of genes and gene products sharing similar GO annotations. The result are shown in an association tree organized according to GO hierarchies and supported with many dynamic display options such as sorting tree nodes or changing orientation of the tree. For GO curators and frequent GO users, DynGO provides fast and convenient access to GO annotation data. DynGO is generally applicable to any data set where the records are annotated with GO terms, as illustrated by two examples. Conclusion We have presented a standalone package DynGO that provides functionalities to search and browse GO and its association databases as well as several additional functions such as batch retrieval and semantic retrieval. The complete documentation and software are

Production of 2-ketoisocaproate with Corynebacterium glutamicum strains devoid of plasmids and heterologous genes.

PubMed

Vogt, Michael; Haas, Sabine; Polen, Tino; van Ooyen, Jan; Bott, Michael

2015-03-01

2-Ketoisocaproate (KIC), the last intermediate in l-leucine biosynthesis, has various medical and industrial applications. After deletion of the ilvE gene for transaminase B in l-leucine production strains of Corynebacterium glutamicum, KIC became the major product, however, the strains were auxotrophic for l-isoleucine. To avoid auxotrophy, reduction of IlvE activity by exchanging the ATG start codon of ilvE by GTG was tested instead of an ilvE deletion. The resulting strains were indeed able to grow in glucose minimal medium without amino acid supplementation, but at the cost of lowered growth rates and KIC production parameters. The best production performance was obtained with strain MV-KICF1, which carried besides the ilvE start codon exchange three copies of a gene for a feedback-resistant 2-isopropylmalate synthase, one copy of a gene for a feedback-resistant acetohydroxyacid synthase and deletions of ltbR and iolR encoding transcriptional regulators. In the presence of 1 mM l-isoleucine, MV-KICF1 accumulated 47 mM KIC (6.1 g l(-1)) with a yield of 0.20 mol/mol glucose and a volumetric productivity of 1.41 mmol KIC l(-1)  h(-1). Since MV-KICF1 is plasmid free and lacks heterologous genes, it is an interesting strain for industrial application and as platform for the production of KIC-derived compounds, such as 3-methyl-1-butanol. © 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

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

PubMed

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

A Weighted Multipath Measurement Based on Gene Ontology for Estimating Gene Products Similarity

PubMed Central

Liu, Lizhen; Dai, Xuemin; Song, Wei; Lu, Jingli

2014-01-01

Abstract Many different methods have been proposed for calculating the semantic similarity of term pairs based on gene ontology (GO). Most existing methods are based on information content (IC), and the methods based on IC are used more commonly than those based on the structure of GO. However, most IC-based methods not only fail to handle identical annotations but also show a strong bias toward well-annotated proteins. We propose a new method called weighted multipath measurement (WMM) for estimating the semantic similarity of gene products based on the structure of the GO. We not only considered the contribution of every path between two GO terms but also took the depth of the lowest common ancestors into account. We assigned different weights for different kinds of edges in GO graph. The similarity values calculated by WMM can be reused because they are only relative to the characteristics of GO terms. Experimental results showed that the similarity values obtained by WMM have a higher accuracy. We compared the performance of WMM with that of other methods using GO data and gene annotation datasets for yeast and humans downloaded from the GO database. We found that WMM is more suited for prediction of gene function than most existing IC-based methods and that it can distinguish proteins with identical annotations (two proteins are annotated with the same terms) from each other. PMID:25229994

Gene expression profiling defined pathways correlated with fibroblast cell proliferation induced by Opisthorchis viverrini excretory/secretory product.

PubMed

Thuwajit, Chanitra; Thuwajit, Peti; Uchida, Kazuhiko; Daorueang, Daoyot; Kaewkes, Sasithorn; Wongkham, Sopit; Miwa, Masanao

2006-06-14

To investigate the mechanism of fibroblast cell proliferation stimulated by the Opisthorchis viverrini excretory/secretory (ES) product. NIH-3T3, mouse fibroblast cells were treated with O. viverrini ES product by non-contact co-cultured with the adult parasites. Total RNA from NIH-3T3 treated and untreated with O. viverrini was extracted, reverse transcribed and hybridized with the mouse 15K complementary DNA (cDNA) array. The result was analyzed by ArrayVision version 5 and GeneSpring version 5 softwares. After normalization, the ratios of gene expression of parasite treated to untreated NIH-3T3 cells of 2-and more-fold upregulated was defined as the differentially expressed genes. The expression levels of the signal transduction genes were validated by semi-quantitative SYBR-based real-time RT-PCR. Among a total of 15,000 genes/ESTs, 239 genes with established cell proliferation-related function were 2 fold-and more-up-regulated by O. viverrini ES product compared to those in cells without exposure to the parasitic product. These genes were classified into groups including energy and metabolism, signal transduction, protein synthesis and translation, matrix and structural protein, transcription control, cell cycle and DNA replication. Moreover, the expressions of serine-threonine kinase receptor, receptor tyrosine kinase and collagen production-related genes were up-regulated by O. viverrini ES product. The expression level of signal transduction genes; pkC, pdgfr alpha, jak 1, eps 8, tgf beta 1i4, strap and h ras measured by real-time RT-PCR confirmed their expression levels to those obtained from cDNA array. However, only the up-regulated expression of pkC, eps 8 and tgfbeta 1i4 which are the downstream signaling molecules of either epidermal growth factor (EGF) or transforming growth factor-beta (TGF-beta) showed statistical significance (P < 0.05). O. viverrini ES product stimulates the significant changes of gene expression in several functional categories

Cloning and characterization of the gene for an additional extracellular serine protease of Bacillus subtilis.

PubMed

Sloma, A; Rufo, G A; Theriault, K A; Dwyer, M; Wilson, S W; Pero, J

1991-11-01

We have purified a minor extracellular serine protease from a strain of Bacillus subtilis bearing null mutations in five extracellular protease genes: apr, npr, epr, bpr, and mpr (A. Sloma, C. Rudolph, G. Rufo, Jr., B. Sullivan, K. Theriault, D. Ally, and J. Pero, J. Bacteriol. 172:1024-1029, 1990). During purification, this novel protease (Vpr) was found bound in a complex in the void volume after gel filtration chromatography. The amino-terminal sequence of the purified protein was determined, and an oligonucleotide probe was constructed on the basis of the amino acid sequence. This probe was used to clone the structural gene (vpr) for this protease. The gene encodes a primary product of 806 amino acids. The amino acid sequence of the mature protein was preceded by a signal sequence of approximately 28 amino acids and a prosequence of approximately 132 amino acids. The mature protein has a predicted molecular weight of 68,197; however, the isolated protein has an apparent molecular weight of 28,500, suggesting that Vpr undergoes C-terminal processing or proteolysis. The vpr gene maps in the ctrA-sacA-epr region of the chromosome and is not required for growth or sporulation.

Production tolerance of additive manufactured polymeric objects for clinical applications.

PubMed

Braian, Michael; Jimbo, Ryo; Wennerberg, Ann

2016-07-01

To determine the production tolerance of four commercially available additive manufacturing systems. By reverse engineering annex A and B from the ISO_12836;2012, two geometrical figures relevant to dentistry was obtained. Object A specifies the measurement of an inlay-shaped object and B a multi-unit specimen to simulate a four-unit bridge model. The objects were divided into x, y and z measurements, object A was divided into a total of 16 parameters and object B was tested for 12 parameters. The objects were designed digitally and manufactured by professionals in four different additive manufacturing systems; each system produced 10 samples of each objects. For object A, three manufacturers presented an accuracy of <100μm and one system showed an accuracy of <20μm. For object B, all systems presented an accuracy of <100μm, and most parameters were <40μm. The standard deviation for most parameters were <40μm. The growing interest and use of intra-oral digitizing systems stresses the use of computer aided manufacturing of working models. The additive manufacturing techniques has the potential to help us in the digital workflow. Thus, it is important to have knowledge about production accuracy and tolerances. This study presents a method to test additive manufacturing units for accuracy and repeatability. Copyright © 2016 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Regulation of docosahexaenoic acid production by Schizochytrium sp.: effect of nitrogen addition.

PubMed

Ren, Lu-Jing; Sun, Li-Na; Zhuang, Xiao-Yan; Qu, Liang; Ji, Xiao-Jun; Huang, He

2014-05-01

Docosahexaenoic acid (DHA) percentage in total fatty acids (TFAs) is an important index in DHA microbial production. In this study, the change of DHA percentage in response to fermentation stages and the strategies to increase DHA percentage were investigated. Two kinds of conventional nitrogen sources, monosodium glutamate (MSG) and ammonium sulfate (AS), were tested to regulate DHA synthesis. Results showed that MSG addition could accelerate the substrate consumption rate but inhibit lipid accumulation, while AS addition could increase DHA percentage in TFAs effectively but extend fermentation period slightly. Finally, the AS addition strategy was successfully applied in 7,000-L fermentor and DHA percentage in TFAs and DHA yield reached 46.06 % and 18.48 g/L, which was 19.54 and 17.41 % higher than that of no-addition strategy. This would provide guidance for the large-scale production of the other similar polyunsaturated fatty acid, and give insight into the nitrogen metabolism in oil-producing microorganisms.

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

DOEpatents

Wohlbach, Dana J.; Gasch, Audrey P.

2015-09-29

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

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

DOEpatents

Wohlbach, Dana J.; Gasch, Audrey P.

2016-11-29

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

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

DOEpatents

Wohlbach, Dana J.; Gasch, Audrey P.

2014-08-05

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

New Japanese Regulatory Frameworks for Clinical Research and Marketing Authorization of Gene Therapy and Cellular Therapy Products.

PubMed

Nagai, Sumimasa; Ozawa, Keiya

2017-01-01

In Japan, the Pharmaceuticals and Medical Devices Law was passed in 2014. In this new law, regenerative medical products were defined, and a conditional and term-limited approval system only for regenerative medical products was instituted. Therefore, regenerative medical products can be approved based on phase I and/or II trials. Gene therapy and adoptive cellular therapy are categorized as regenerative medical products. This law is intended for registration trials for marketing authorization. The Act on the Safety of Regenerative Medicine was also implemented in 2014. This act is intended for clinical research and medical practice involving processed cells other than registration trials. Under this act, a review of plans on medical treatments or clinical studies by a certified committee and submission of the plans to the Ministry of Health, Labour and Welfare (MHLW) are mandatory. The MHLW instituted the SAKIGAKE (meaning a pioneer or forerunner in Japanese) designation system in 2015. This designation is similar to the breakthrough therapy designation in the US and PRIME in the EU. In addition, the MHLW started the "Project for Enhanced Practical Application of Innovative Drugs, Medical Devices and Regenerative Medical Products" to promote personnel exchange and cooperation in writing of guidelines on the evaluation of innovative medical products between the Pharmaceuticals and Medical Devices Agency and academia. Some new guidelines regarding gene and cellular therapy were published. In this review, we comprehensively described these complicated regulations and problems to be solved in order to facilitate global readers' understanding of Japanese regulatory frameworks. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Clinical trials of GMP products in the gene therapy field.

PubMed

Bamford, Kathleen B

2011-01-01

Advances in gene therapy are increasingly leading to clinical assessment in many fields of medicine with diverse approaches. The basic science stems from approaches aimed at different functions such as correcting a missing/abnormal gene, altering the proportion or expression of normal genes to augment a physiological process or using this principle to destroy malignant or infected cells. As the technology advances, it is increasingly important to ensure that clinical trials answer the questions that need to be asked. In this chapter we review examples of published clinical trials, resources for accessing information about registered trials, the process of regulating trials, good clinical practice, and good manufacturing practice as well as summarising the approach taken by regulatory authorities in reviewing applications for the introduction of products for use in the clinic.

Additive Manufactured Product Integrity

NASA Technical Reports Server (NTRS)

Waller, Jess; Wells, Doug; James, Steve; Nichols, Charles

2017-01-01

NASA is providing key leadership in an international effort linking NASA and non-NASA resources to speed adoption of additive manufacturing (AM) to meet NASA's mission goals. Participants include industry, NASA's space partners, other government agencies, standards organizations and academia. Nondestructive Evaluation (NDE) is identified as a universal need for all aspects of additive manufacturing.

Production of Recombinant Adenovirus Containing Human Interlukin-4 Gene

PubMed Central

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 that made it a good choice for using in cancer gene therapy, controlling inflammatory diseases, and studies on autoimmune diseases. Materials and Methods In brief, IL-4 coding sequence was amplified by and cloned in pAd-Track-CMV. Then, by means of homologous recombination between recombinant pAd-Track-CMV and Adeasy-1 plasmid in bacteria, recombinant adenovirus complete genome was made and IL-4 containing shuttle vector was incorporated into the viral backbone. After linearization, for virus packaging, viral genome was transfected into HEK-293 cell line. Viral production was conveniently followed with the aid of green fluorescent protein. Results Recombinant adenovirus produced here, was capable to infecting cell lines and express interlukin-4 in cell. Conclusion This system can be used as a powerful, easy, and cost benefit tool in various studies on cancer gene therapy and also studies on immunogenetics. PMID:23493491

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

PubMed

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.

An additional k-means clustering step improves the biological features of WGCNA gene co-expression networks.

PubMed

Botía, Juan A; Vandrovcova, Jana; Forabosco, Paola; Guelfi, Sebastian; D'Sa, Karishma; Hardy, John; Lewis, Cathryn M; Ryten, Mina; Weale, Michael E

2017-04-12

Weighted Gene Co-expression Network Analysis (WGCNA) is a widely used R software package for the generation of gene co-expression networks (GCN). WGCNA generates both a GCN and a derived partitioning of clusters of genes (modules). We propose k-means clustering as an additional processing step to conventional WGCNA, which we have implemented in the R package km2gcn (k-means to gene co-expression network, https://github.com/juanbot/km2gcn ). We assessed our method on networks created from UKBEC data (10 different human brain tissues), on networks created from GTEx data (42 human tissues, including 13 brain tissues), and on simulated networks derived from GTEx data. We observed substantially improved module properties, including: (1) few or zero misplaced genes; (2) increased counts of replicable clusters in alternate tissues (x3.1 on average); (3) improved enrichment of Gene Ontology terms (seen in 48/52 GCNs) (4) improved cell type enrichment signals (seen in 21/23 brain GCNs); and (5) more accurate partitions in simulated data according to a range of similarity indices. The results obtained from our investigations indicate that our k-means method, applied as an adjunct to standard WGCNA, results in better network partitions. These improved partitions enable more fruitful downstream analyses, as gene modules are more biologically meaningful.

Improved pestalotiollide B production by deleting competing polyketide synthase genes in Pestalotiopsis microspora.

PubMed

Chen, Longfei; Li, Yingying; Zhang, Qian; Wang, Dan; Akhberdi, Oren; Wei, Dongsheng; Pan, Jiao; Zhu, Xudong

2017-02-01

Pestalotiollide B, an analog of dibenzodioxocinones which are inhibitors of cholesterol ester transfer proteins, is produced by Pestalotiopsis microspora NK17. To increase the production of pestalotiollide B, we attempted to eliminate competing polyketide products by deleting the genes responsible for their biosynthesis. We successfully deleted 41 out of 48 putative polyketide synthases (PKSs) in the genome of NK17. Nine of the 41 PKS deleted strains had significant increased production of pestalotiollide B (P < 0.05). For instance, deletion of pks35, led to an increase of pestalotiollide B by 887%. We inferred that these nine PKSs possibly lead to branch pathways that compete for precursors with pestalotiollide B, or that convert the product. Deletion of some other PKS genes such as pks8 led to a significant decrease of pestalotiollide B, suggesting they are responsible for its biosynthesis. Our data demonstrated that improvement of pestalotiollide B production can be achieved by eliminating competing polyketides.

Overexpression of the gene encoding HMG-CoA reductase in Saccharomyces cerevisiae for production of prenyl alcohols.

PubMed

Ohto, Chikara; Muramatsu, Masayoshi; Obata, Shusei; Sakuradani, Eiji; Shimizu, Sakayu

2009-04-01

To develop microbial production method for prenyl alcohols (e.g., (E,E)-farnesol (FOH), (E)-nerolidol (NOH), and (E,E,E)-geranylgeraniol (GGOH)), the genes encoding enzymes in the mevalonate and prenyl diphosphate pathways were overexpressed in Saccharomyces cerevisiae, and the resultant transformants were evaluated as to the production of these alcohols. Overexpression of the gene encoding hydroxymethylglutaryl (HMG)-CoA reductase was most effective among the genes tested. A derivative of S. cerevisiae ATCC 200589, which was selected through screening, was found to be the most suitable host for the production. On cultivation of the resultant transformant, in which the HMG-CoA reductase gene was overexpressed, in a 5-liter bench-scale jar fermenter for 7 d, the production of FOH, NOH, and GGOH reached 145.7, 98.8, and 2.46 mg/l, respectively.

Ethanol production by recombinant hosts

DOEpatents

Fowler, David E.; Horton, Philip G.; Ben-Bassat, Arie

1996-01-01

Novel plasmids comprising genes which code for the alcohol dehydrogenase and pyruvate decarboxylase are described. Also described are recombinant hosts which have been transformed with genes coding for alcohol dehydrogenase and pyruvate. By virtue of their transformation with these genes, the recombinant hosts are capable of producing significant amounts of ethanol as a fermentation product. Also disclosed are methods for increasing the growth of recombinant hosts and methods for reducing the accumulation of undesirable metabolic products in the growth medium of these hosts. Also disclosed are recombinant host capable of producing significant amounts of ethanol as a fermentation product of oligosaccharides and plasmids comprising genes encoding polysaccharases, in addition to the genes described above which code for the alcohol dehydrogenase and pyruvate decarboxylase. Further, methods are described for producing ethanol from oligomeric feedstock using the recombinant hosts described above. Also provided is a method for enhancing the production of functional proteins in a recombinant host comprising overexpressing an adhB gene in the host. Further provided are process designs for fermenting oligosaccharide-containing biomass to ethanol.

Ethanol production by recombinant hosts

DOEpatents

Ingram, Lonnie O.; Beall, David S.; Burchhardt, Gerhard F. H.; Guimaraes, Walter V.; Ohta, Kazuyoshi; Wood, Brent E.; Shanmugam, Keelnatham T.

1995-01-01

Novel plasmids comprising genes which code for the alcohol dehydrogenase and pyruvate decarboxylase are described. Also described are recombinant hosts which have been transformed with genes coding for alcohol dehydrogenase and pyruvate. By virtue of their transformation with these genes, the recombinant hosts are capable of producing significant amounts of ethanol as a fermentation product. Also disclosed are methods for increasing the growth of recombinant hosts and methods for reducing the accumulation of undesirable metabolic products in the growth medium of these hosts. Also disclosed are recombinant host capable of producing significant amounts of ethanol as a fermentation product of oligosaccharides and plasmids comprising genes encoding polysaccharases, in addition to the genes described above which code for the alcohol dehydrogenase and pyruvate decarboxylase. Further, methods are described for producing ethanol from oligomeric feedstock using the recombinant hosts described above. Also provided is a method for enhancing the production of functional proteins in a recombinant host comprising overexpressing an adhB gene in the host. Further provided are process designs for fermenting oligosaccharide-containing biomass to ethanol.

Resistance gene candidates identified by PCR with degenerate oligonucleotide primers map to clusters of resistance genes in lettuce.

PubMed

Shen, K A; Meyers, B C; Islam-Faridi, M N; Chin, D B; Stelly, D M; Michelmore, R W

1998-08-01

The recent cloning of genes for resistance against diverse pathogens from a variety of plants has revealed that many share conserved sequence motifs. This provides the possibility of isolating numerous additional resistance genes by polymerase chain reaction (PCR) with degenerate oligonucleotide primers. We amplified resistance gene candidates (RGCs) from lettuce with multiple combinations of primers with low degeneracy designed from motifs in the nucleotide binding sites (NBSs) of RPS2 of Arabidopsis thaliana and N of tobacco. Genomic DNA, cDNA, and bacterial artificial chromosome (BAC) clones were successfully used as templates. Four families of sequences were identified that had the same similarity to each other as to resistance genes from other species. The relationship of the amplified products to resistance genes was evaluated by several sequence and genetic criteria. The amplified products contained open reading frames with additional sequences characteristic of NBSs. Hybridization of RGCs to genomic DNA and to BAC clones revealed large numbers of related sequences. Genetic analysis demonstrated the existence of clustered multigene families for each of the four RGC sequences. This parallels classical genetic data on clustering of disease resistance genes. Two of the four families mapped to known clusters of resistance genes; these two families were therefore studied in greater detail. Additional evidence that these RGCs could be resistance genes was gained by the identification of leucine-rich repeat (LRR) regions in sequences adjoining the NBS similar to those in RPM1 and RPS2 of A. thaliana. Fluorescent in situ hybridization confirmed the clustered genomic distribution of these sequences. The use of PCR with degenerate oligonucleotide primers is therefore an efficient method to identify numerous RGCs in plants.

Genetic resources for methane production from biomass described with the Gene Ontology.

PubMed

Purwantini, Endang; Torto-Alalibo, Trudy; Lomax, Jane; Setubal, João C; Tyler, Brett M; Mukhopadhyay, Biswarup

2014-01-01

Methane (CH4) is a valuable fuel, constituting 70-95% of natural gas, and a potent greenhouse gas. Release of CH4 into the atmosphere contributes to climate change. Biological CH4 production or methanogenesis is mostly performed by methanogens, a group of strictly anaerobic archaea. The direct substrates for methanogenesis are H2 plus CO2, acetate, formate, methylamines, methanol, methyl sulfides, and ethanol or a secondary alcohol plus CO2. In numerous anaerobic niches in nature, methanogenesis facilitates mineralization of complex biopolymers such as carbohydrates, lipids and proteins generated by primary producers. Thus, methanogens are critical players in the global carbon cycle. The same process is used in anaerobic treatment of municipal, industrial and agricultural wastes, reducing the biological pollutants in the wastes and generating methane. It also holds potential for commercial production of natural gas from renewable resources. This process operates in digestive systems of many animals, including cattle, and humans. In contrast, in deep-sea hydrothermal vents methanogenesis is a primary production process, allowing chemosynthesis of biomaterials from H2 plus CO2. In this report we present Gene Ontology (GO) terms that can be used to describe processes, functions and cellular components involved in methanogenic biodegradation and biosynthesis of specialized coenzymes that methanogens use. Some of these GO terms were previously available and the rest were generated in our Microbial Energy Gene Ontology (MENGO) project. A recently discovered non-canonical CH4 production process is also described. We have performed manual GO annotation of selected methanogenesis genes, based on experimental evidence, providing "gold standards" for machine annotation and automated discovery of methanogenesis genes or systems in diverse genomes. Most of the GO-related information presented in this report is available at the MENGO website (http://www.mengo.biochem.vt.edu/).

Genetic resources for methane production from biomass described with the Gene Ontology

PubMed Central

Purwantini, Endang; Torto-Alalibo, Trudy; Lomax, Jane; Setubal, João C.; Tyler, Brett M.; Mukhopadhyay, Biswarup

2014-01-01

Methane (CH4) is a valuable fuel, constituting 70–95% of natural gas, and a potent greenhouse gas. Release of CH4 into the atmosphere contributes to climate change. Biological CH4 production or methanogenesis is mostly performed by methanogens, a group of strictly anaerobic archaea. The direct substrates for methanogenesis are H2 plus CO2, acetate, formate, methylamines, methanol, methyl sulfides, and ethanol or a secondary alcohol plus CO2. In numerous anaerobic niches in nature, methanogenesis facilitates mineralization of complex biopolymers such as carbohydrates, lipids and proteins generated by primary producers. Thus, methanogens are critical players in the global carbon cycle. The same process is used in anaerobic treatment of municipal, industrial and agricultural wastes, reducing the biological pollutants in the wastes and generating methane. It also holds potential for commercial production of natural gas from renewable resources. This process operates in digestive systems of many animals, including cattle, and humans. In contrast, in deep-sea hydrothermal vents methanogenesis is a primary production process, allowing chemosynthesis of biomaterials from H2 plus CO2. In this report we present Gene Ontology (GO) terms that can be used to describe processes, functions and cellular components involved in methanogenic biodegradation and biosynthesis of specialized coenzymes that methanogens use. Some of these GO terms were previously available and the rest were generated in our Microbial Energy Gene Ontology (MENGO) project. A recently discovered non-canonical CH4 production process is also described. We have performed manual GO annotation of selected methanogenesis genes, based on experimental evidence, providing “gold standards” for machine annotation and automated discovery of methanogenesis genes or systems in diverse genomes. Most of the GO-related information presented in this report is available at the MENGO website (http

Characterization of Clostridium perfringens TpeL Toxin Gene Carriage, Production, Cytotoxic Contributions, and Trypsin Sensitivity

PubMed Central

Chen, Jianming

2015-01-01

Large clostridial toxins (LCTs) are produced by at least four pathogenic clostridial species, and several LCTs are proven pivotal virulence factors for both human and veterinary diseases. TpeL is a recently identified LCT produced by Clostridium perfringens that has received relatively limited study. In response, the current study surveyed carriage of the tpeL gene among different C. perfringens strains, detecting this toxin gene in some type A, B, and C strains but not in any type D or E strains. This study also determined that all tested strains maximally produce, and extracellularly release, TpeL at the late-log or early-stationary growth stage during in vitro culture, which is different from the maximal late-stationary-phase production reported previously for other LCTs and for TpeL production by C. perfringens strain JIR12688. In addition, the present study found that TpeL levels in culture supernatants can be repressed by either glucose or sucrose. It was also shown that, at natural production levels, TpeL is a significant contributor to the cytotoxic activity of supernatants from cultures of tpeL-positive strain CN3685. Lastly, this study identified TpeL, which presumably is produced in the intestines during diseases caused by TpeL-positive type B and C strains, as a toxin whose cytotoxicity decreases after treatment with trypsin; this finding may have pathophysiologic relevance by suggesting that, like beta toxin, TpeL contributes to type B and C infections in hosts with decreased trypsin levels due to disease, diet, or age. PMID:25824828

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

PubMed

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.

Isolated Fungal Promoters and Gene Transcription Terminators and Methods of Protein and Chemical Production in a Fungus

DOEpatents

Dai, Ziyu; Lasure, Linda L.; Magnuson, Jon K.

2008-11-11

The present invention encompasses isolated gene regulatory elements and gene transcription terminators that are differentially expressed in a native fungus exhibiting a first morphology relative to the native fungus exhibiting a second morphology. The invention also encompasses a method of utilizing a fungus for protein or chemical production. A transformed fungus is produced by transforming a fungus with a recombinant polynucleotide molecule. The recombinant polynucleotide molecule contains an isolated polynucleotide sequence linked operably to another molecule comprising a coding region of a gene of interest. The gene regulatory element and gene transcription terminator may temporally and spatially regulate expression of particular genes for optimum production of compounds of interest in a transgenic fungus.

Isolated fungal promoters and gene transcription terminators and methods of protein and chemical production in a fungus

DOEpatents

Dai, Ziyu; Lasure, Linda L.; Magnuson, Jon K.

2008-11-11

The present invention encompasses isolated gene regulatory elements and gene transcription terminators that are differentially expressed in a native fungus exhibiting a first morphology relative to the native fungus exhibiting a second morphology. The invention also encompasses a method of utilizing a fungus for protein or chemical production. A transformed fungus is produced by transforming a fungus with a recombinant polynucleotide molecule. The recombinant polynucleotide molecule contains an isolated polynucleotide sequence linked operably to another molecule comprising a coding region of a gene of interest. The gene regulatory element and gene transcription terminator may temporally and spatially regulate expression of particular genes for optimum production of compounds of interest in a transgenic fungus.

Isolated fungal promoters and gene transcription terminators and methods of protein and chemical production in a fungus

DOEpatents

Dai, Ziyu; Lasure, Linda L; Magnuson, Jon K

2014-05-27

The present invention encompasses isolated gene regulatory elements and gene transcription terminators that are differentially expressed in a native fungus exhibiting a first morphology relative to the native fungus exhibiting a second morphology. The invention also encompasses a method of utilizing a fungus for protein or chemical production. A transformed fungus is produced by transforming a fungus with a recombinant polynucleotide molecule. The recombinant polynucleotide molecule contains an isolated polynucleotide sequence linked operably to another molecule comprising a coding region of a gene of interest. The gene regulatory element and gene transcription terminator may temporally and spatially regulate expression of particular genes for optimum production of compounds of interest in a transgenic fungus.

Tip-alpha (hp0596 gene product) is a highly immunogenic Helicobacter pylori protein involved in colonization of mouse gastric mucosa.

PubMed

Godlewska, Renata; Pawlowski, Marcin; Dzwonek, Artur; Mikula, Michal; Ostrowski, Jerzy; Drela, Nadzieja; Jagusztyn-Krynicka, Elzbieta K

2008-03-01

A product of the Helicobacter pylori hp0596 gene (Tip-alpha) is a highly immunogenic homodimeric protein, unique for this bacterium. Cell fractionation experiments indicate that Tip-alpha is anchored to the inner membrane. In contrast, the three-dimensional model of the protein suggests that Tip-alpha is soluble or, at least, largely exposed to the solvent. hp0596 gene knockout resulted in a significant decrease in the level of H. pylori colonization as measured by real-time PCR assay. In addition, the Tip-alpha recombinant protein was determined to stimulate macrophage to produce IL-1alpha and TNF-alpha. Both results imply that Tip-alpha is rather loosely connected to the inner membrane and potentially released during infection.

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.

PubMed

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.

An Introductory Bioinformatics Exercise to Reinforce Gene Structure and Expression and Analyze the Relationship between Gene and Protein Sequences

ERIC Educational Resources Information Center

Almeida, Craig A.; Tardiff, Daniel F.; De Luca, Jane P.

2004-01-01

We have developed an introductory bioinformatics exercise for sophomore biology and biochemistry students that reinforces the understanding of the structure of a gene and the principles and events involved in its expression. In addition, the activity illustrates the severe effect mutations in a gene sequence can have on the protein product.…

15 CFR 714.3 - Advance declaration requirements for additionally planned production of Schedule 3 chemicals.

Code of Federal Regulations, 2011 CFR

2011-01-01

... COMMERCE CHEMICAL WEAPONS CONVENTION REGULATIONS ACTIVITIES INVOLVING SCHEDULE 3 CHEMICALS § 714.3 Advance declaration requirements for additionally planned production of Schedule 3 chemicals. (a) Declaration... additionally planned production of Schedule 3 chemicals. 714.3 Section 714.3 Commerce and Foreign Trade...

Streptomyces associated with a marine sponge Haliclona sp.; biosynthetic genes for secondary metabolites and products.

PubMed

Khan, Shams Tabrez; Komaki, Hisayuki; Motohashi, Keiichiro; Kozone, Ikuko; Mukai, Akira; Takagi, Motoki; Shin-ya, Kazuo

2011-02-01

Terrestrial actinobacteria have served as a primary source of bioactive compounds; however, a rapid decrease in the discovery of new compounds strongly necessitates new investigational approaches. One approach is the screening of actinobacteria from marine habitats, especially the members of the genus Streptomyces. Presence of this genus in a marine sponge, Haliclona sp., was investigated using culture-dependent and -independent techniques. 16S rRNA gene clone library analysis showed the presence of diverse Streptomyces in the sponge sample. In addition to the dominant genus Streptomyces, members of six different genera were isolated using four different media. Five phylogenetically new strains, each representing a novel species in the genus Streptomyces were also isolated. Polyphasic study suggesting the classification of two of these strains as novel species is presented. Searching the strains for the production of novel compounds and the presence of biosynthetic genes for secondary metabolites revealed seven novel compounds and biosynthetic genes with unique sequences. In these compounds, JBIR-43 exhibited cytotoxic activity against cancer cell lines. JBIR-34 and -35 were particularly interesting because of their unique chemical skeleton. To our knowledge, this is the first comprehensive study detailing the isolation of actinobacteria from a marine sponge and novel secondary metabolites from these strains.

Production of Dwarf Lettuce by Overexpressing a Pumpkin Gibberellin 20-Oxidase Gene

PubMed Central

Niki, Tomoya; Nishijima, Takaaki; Nakayama, Masayoshi; Hisamatsu, Tamotsu; Oyama-Okubo, Naomi; Yamazaki, Hiroko; Hedden, Peter; Lange, Theo; Mander, Lewis N.; Koshioka, Masaji

2001-01-01

We investigated the effect of overexpressing a pumpkin gibberellin (GA) 20-oxidase gene encoding an enzyme that forms predominantly biologically inactive products on GA biosynthesis and plant morphology in transgenic lettuce (Lactuca sativa cv Vanguard) plants. Lettuce was transformed with the pumpkin GA 20-oxidase gene downstream of a strong constitutive promoter cassette (El2–35S-Ω). The transgenic plants in which the pumpkin gene was detected by polymerase chain reaction were dwarfed in the T2 generation, whereas transformants with a normal growth phenotype did not contain the transgene. The result of Southern-blot analysis showed that the transgene was integrated as a single copy; the plants segregated three dwarfs to one normal in the T2 generation, indicating that the transgene was stable and dominant. The endogenous levels of GA1 and GA4 were reduced in the dwarfs, whereas large amounts of GA17 and GA25, which are inactive products of the pumpkin GA 20-oxidase, accumulated in these lines. These results indicate that a functional pumpkin GA 20-oxidase is expressed in the transgenic lettuce, resulting in a diversion of the normal pathway of GA biosynthesis to inactive products. Furthermore, this technique may be useful for controlling plant stature in other agricultural and horticultural species. PMID:11457947

mcrA Gene abundance correlates with hydrogenotrophic methane production rates in full-scale anaerobic waste treatment systems.

PubMed

Morris, R L; Tale, V P; Mathai, P P; Zitomer, D H; Maki, J S

2016-02-01

Anaerobic treatment is a sustainable and economical technology for waste stabilization and production of methane as a renewable energy. However, the process is under-utilized due to operational challenges. Organic overload or toxicants can stress the microbial community that performs waste degradation, resulting in system failure. In addition, not all methanogenic microbial communities are equally capable of consistent, maximum biogas production. Opinion varies as to which parameters should be used to monitor the fitness of digester biomass. No standard molecular tools are currently in use to monitor and compare full-scale operations. It was hypothesized that determining the number of gene copies of mcrA, a methanogen-specific gene, would positively correlate with specific methanogenic activity (SMA) rates from biomass samples from six full-scale anaerobic digester systems. Positive correlations were observed between mcrA gene copy numbers and methane production rates against H2  : CO2 and propionate (R(2)  = 0·67-0·70, P < 0·05) but not acetate (R(2)  = 0·49, P > 0·05). Results from this study indicate that mcrA gene targeted qPCR can be used as an alternate tool to monitor and compare certain methanogen communities in anaerobic digesters. Using quantitative PCR (qPCR), we demonstrate that the abundance of mcrA, a gene specific to methane producing archaea, correlated with specific methanogenic activity (SMA) measurements when H2 and CO2 , or propionate were provided as substrates. However, mcrA abundance did not correlate with SMA with acetate. SMA values are often used as a fitness indicator of anaerobic biomass. Results from qPCR can be obtained within a day while SMA analysis requires days to weeks to complete. Therefore, qPCR for mcrA abundance is a sensitive and fast method to compare and monitor the fitness of certain anaerobic biomass. As a monitoring tool, qPCR of mcrA will help anaerobic digester operators optimize treatment and encourage

Impact of trace element addition on biogas production from food industrial waste--linking process to microbial communities.

PubMed

Feng, Xin Mei; Karlsson, Anna; Svensson, Bo H; Bertilsson, Stefan

2010-10-01

Laboratory-scale reactors treating food industry waste were used to investigate the effects of additions of cobalt (Co), nickel/molybdenum/boron (Ni/Mo/B) and selenium/tungsten (Se/W) on the biogas process and the associated microbial community. The highest methane production (predicted value: 860 mL g(-1) VS) was linked to high Se/W concentrations in combination with a low level of Co. A combination of quantitative real-time PCR of 16S rRNA genes, terminal restriction fragment length polymorphism (T-RFLP) and clone library sequencing was used for the community analysis. The T-RFLP data show a higher diversity for bacteria than for archaea in all the treatments. The most abundant bacterial population (31-55% of the total T-RFLP fragments' intensity) was most closely related to Actinomyces europaeus (94% homology). Two dominant archaeal populations shared 98-99% sequence homology with Methanosarcina siciliae and Methanoculleus bourgensis, respectively. Only limited influence of the trace metal additions was found on the bacterial community composition, with two bacterial populations responding to the addition of a combination of Ni/Mo/B, while the dominant archaeal populations were influenced by the addition of Ni/Mo/B and/or Se/W. The maintenance of methanogenic activity was largely independent of archaeal community composition, suggesting a high degree of functional redundancy in the methanogens of the biogas reactors. © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Effect of the addition of fatty by-products from the refining of vegetable oil on methane production in co-digestion.

PubMed

Torrijos, M; Sousbie, P; Badey, L; Bosque, F; Steyer, J P

2012-01-01

The purpose of this work was to investigate the effects of the addition of by-products from the refining of vegetable oil on the behavior of co-digestion reactors treating a mixture of grass, cow dung and fruit and vegetable waste. Three by-products were used: one soapstock, one used winterization earth and one skimming of aeroflotation of the effluents. Three 15 l reactors were run in parallel and fed five times a week. In a first phase of 4 weeks, the three reactors were fed with the co-digestion substrates alone (grass, cow dung and fruit and vegetable waste) at an organic loading rate (OLR) of 1.5 g VS/kg d (VS: volatile solids). Then, a different by-product from the refining of oil was added to the feed of each reactor at an OLR of 0.5 g VS/kg d, generating a 33% increase in the OLR. The results show that the addition of by-products from the refining of oil is an efficient way of increasing the methane production of co-digestion reactors thanks to high methane yield of such by-products (0.69-0.77 l CH(4)/g VS loaded). In fact, in this work, it was possible to raise the methane production of the reactors by about 60% through a 33% increase in the OLR thanks to the addition of the by-products from the refining of vegetable oil.

Emerging role of phenolic compounds as natural food additives in fish and fish products.

PubMed

Maqsood, Sajid; Benjakul, Soottawat; Shahidi, Fereidoon

2013-01-01

Chemical and microbiological deteriorations are principal causes of quality loss of fish and fish products during handling, processing, and storage. Development of rancid odor and unpleasant flavor, changes of color and texture as well as lowering nutritional value in fish can be prevented by appropriate use of additives. Due to the potential health hazards of synthetic additives, natural products, especially antioxidants and antimicrobial agents, have been intensively examined as safe alternatives to synthetic compounds. Polyphenols (PP) are the natural antioxidants prevalent in fruits, vegetables, beverages (tea, wine, juices), plants, seaweeds, and some herbs and show antioxidative and antimicrobial activities in different fish and fish products. The use of phenolic compounds also appears to be a good alternative for sulphiting agent for retarding melanosis in crustaceans. Phenolic compounds have also been successfully employed as the processing aid for texture modification of fish mince and surimi. Thus, plant polyphenolic compounds can serve as potential additives for preventing quality deterioration or to retain the quality of fish and fish products.

Cloning and characterization of the gene for an additional extracellular serine protease of Bacillus subtilis.

PubMed Central

Sloma, A; Rufo, G A; Theriault, K A; Dwyer, M; Wilson, S W; Pero, J

1991-01-01

We have purified a minor extracellular serine protease from a strain of Bacillus subtilis bearing null mutations in five extracellular protease genes: apr, npr, epr, bpr, and mpr (A. Sloma, C. Rudolph, G. Rufo, Jr., B. Sullivan, K. Theriault, D. Ally, and J. Pero, J. Bacteriol. 172:1024-1029, 1990). During purification, this novel protease (Vpr) was found bound in a complex in the void volume after gel filtration chromatography. The amino-terminal sequence of the purified protein was determined, and an oligonucleotide probe was constructed on the basis of the amino acid sequence. This probe was used to clone the structural gene (vpr) for this protease. The gene encodes a primary product of 806 amino acids. The amino acid sequence of the mature protein was preceded by a signal sequence of approximately 28 amino acids and a prosequence of approximately 132 amino acids. The mature protein has a predicted molecular weight of 68,197; however, the isolated protein has an apparent molecular weight of 28,500, suggesting that Vpr undergoes C-terminal processing or proteolysis. The vpr gene maps in the ctrA-sacA-epr region of the chromosome and is not required for growth or sporulation. Images FIG. 1 PMID:1938892

High Polyhydroxybutyrate Production in Pseudomonas extremaustralis Is Associated with Differential Expression of Horizontally Acquired and Core Genome Polyhydroxyalkanoate Synthase Genes

PubMed Central

Catone, Mariela V.; Ruiz, Jimena A.; Castellanos, Mildred; Segura, Daniel; Espin, Guadalupe; López, Nancy I.

2014-01-01

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. PMID:24887088

The Role of Cocoa as a Cigarette Additive: Opportunities for Product Regulation

PubMed Central

Kennedy, Ryan David; Connolly, Gregory N.

2014-01-01

Introduction: The 2009 Family Smoking Prevention and Tobacco Control Act prohibited the use of characterizing flavors in cigarettes; however, some of these flavors are still used in cigarettes at varying levels. We reviewed tobacco industry internal documents to investigate the role of one of these flavors, cocoa, with the objective of understanding its relationship to sensory and risk perception, promotion of dependence, and enhancement of attractiveness and acceptability. Methods: We used the Legacy Tobacco Documents Library to identify documents relevant to our research questions. Initial search terms were generated following an examination of published literature on cocoa, other cigarette additives, and sensory and risk perception. Further research questions and search terms were generated based on review of documents generated from the initial search terms. Results: Cocoa is widely applied to cigarettes and has been used by the tobacco industry as an additive since the early 20th century. Cocoa can alter the sensory properties of cigarette smoke, including by providing a more appealing taste and decreasing its harshness. The tobacco industry has experimented with manipulating cocoa levels as a means of achieving sensory properties that appeal to women and youth. Conclusions: Although cocoa is identified as a flavor on tobacco industry Web sites, it may serve other sensory purposes in cigarettes as well. Eliminating cocoa as an additive from tobacco products may affect tobacco product abuse liability by altering smokers’ perceptions of product risk, and decreasing product appeal, especially among vulnerable populations. PMID:24610479

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.

PubMed

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 bacterial ethylene-forming enzyme gene in Trichoderma reesei enhanced the production of ethylene

PubMed Central

Chen, Xi; Liang, Yong; Hua, Jing; Tao, Li; Qin, Wensheng; Chen, Sanfeng

2010-01-01

In order to efficiently utilize natural cellulose materials to produce ethylene, three expression vectors containing the ethylene-forming enzyme (efe) gene from Pseudomonas syringae pv. glycinea were constructed. The target gene was respectively controlled by different promoters: cbh I promoter from Trichoderma reesei cellobiohydrolases I gene, gpd promoter from Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase gene and pgk I promoter from T. reesei 3-phosphoglycerate kinase I gene. After transforming into T. reesei QM9414, 43 stable transformants were obtained by PCR amplification and ethylene determination. Southern blot analysis of 14 transformants demonstrated that the efe gene was integrated into chromosomal DNA with copy numbers from 1 to 4. Reverse transcription polymerase chain reaction (RT-PCR) analysis of 6 transformants showed that the heterologous gene was transcribed. By using wheat straw as a carbon source, the ethylene production rates of aforementioned 14 transformants were measured. Transformant C30-3 with pgk I promoter had the highest ethylene production (4,012 nl h-1 l-1). This indicates that agricultural wastes could be used to produce ethylene in recombinant filamentous fungus T. reesei. PMID:20150979

Dominance and parent-of-origin effects of coding and non-coding alleles at the acylCoA-diacylglycerol-acyltransferase (DGAT1) gene on milk production traits in German Holstein cows

PubMed Central

Kuehn, Christa; Edel, Christian; Weikard, Rosemarie; Thaller, Georg

2007-01-01

Background Substantial gene substitution effects on milk production traits have formerly been reported for alleles at the K232A and the promoter VNTR loci in the bovine acylCoA-diacylglycerol-acyltransferase 1 (DGAT1) gene by using data sets including sires with accumulated phenotypic observations of daughters (breeding values, daughter yield deviations). However, these data sets prevented analyses with respect to dominance or parent-of-origin effects, although an increasing number of reports in the literature outlined the relevance of non-additive gene effects on quantitative traits. Results Based on a data set comprising German Holstein cows with direct trait measurements, we first confirmed the previously reported association of DGAT1 promoter VNTR alleles with milk production traits. We detected a dominant mode of effects for the DGAT1 K232A and promoter VNTR alleles. Namely, the contrasts between the effects of heterozygous individuals at the DGAT1 loci differed significantly from the midpoint between the effects for the two homozygous genotypes for several milk production traits, thus indicating the presence of dominance. Furthermore, we identified differences in the magnitude of effects between paternally and maternally inherited DGAT1 promoter VNTR – K232A haplotypes indicating parent-of-origin effects on milk production traits. Conclusion Non-additive effects like those identified at the bovine DGAT1 locus have to be accounted for in more specific QTL detection models as well as in marker assisted selection schemes. The DGAT1 alleles in cattle will be a useful model for further investigations on the biological background of non-additive effects in mammals due to the magnitude and consistency of their effects on milk production traits. PMID:17892573

The effects of flue gas desulfurization (FGD) system additives on solid by-products. Final report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huyck, K.A.; Tatum, G.S.; DeKraker, D.P.

This study was designed to examine characteristics of by-products from flue gas desulfurization (FGD) systems that use performance-enhancing additives. The by-products were evaluated for handling, disposal, properties, and utilization potential. This study was designed to identify potential operation-limiting consequences (fatal flaws) caused by using FGD system additives. It was not intended to be an all-encompassing study of the possible process, environmental, and utilization circumstances at each plant. Seven utilities conducting additive testing agreed to provide samples for this study both before and during their testing. Solid samples were collected from FGD systems using the following additives: di-basic acid (DBA), whichmore » is a mixture of succinic, glutaric, and adipic acids; formate; sulfur; ethylene diamine tetraacetic acid (EDTA); and chloride. Changes in handling properties that may alter process operations were observed at sites where DBS was used. Changes in leaching behavior, permeability and unconfined compressive strength (properties that affect by-product disposal properties) showed no clear trend for additive or system type. The intent of this study is to provide an overall assessment of potential system-limiting effects of FGD additive use. There were no overriding problems relating to disposal with the use of additives. However, properties of solids produced with and without additives should be tested at each site prior to use of an FGD system additive. This is necessary because of the variability in state disposal and permitting requirements and the influence of specific operating conditions on by-product properties.« less

Improved ethanol production in the presence of cadmium ions by a Saccharomyces cerevisiae transformed with a novel cadmium-resistance gene DvCRP1.

PubMed

Hu, Jiajun; Xu, Qingyun; Wu, Mengnan; Meng, Xiangzong; Song, Rentao; Gao, Mintian

2016-11-01

The DvCRP1 gene obtained from Dunaliella viridis is a cadmium-resistance gene that induces cadmium accumulation in microbial and plant cells. In the present study, Saccharomyces cerevisiae was used as a model system to investigate the effect of DvCRP1 on both cadmium detoxification and ethanol production. Inhibitory effects of cadmium (50-300 µmol/L) on growth (29-92%), glucose consumption (23-89%), and ethanol production (17-92%) were observed at 24 h by S. cerevisiae. DvCRP1 alleviated the inhibitory effect of cadmium, with increase in the ethanol production. The established mathematical model showed that the initial inoculation concentration, cadmium concentration, and transformation of DvCRP1 were the most important factors for cell growth, glucose consumption, and ethanol production. Cadmium detoxification of yeast was also enhanced by increasing the initial concentration of yeast cells. Transforming with DvCRP1 further enhanced detoxification, especially at high cadmium concentrations. Transforming with DvCRP1 further enhanced detoxification, especially at high cadmium concentrations (200 µmol/L). The present results evidenced the potential of the insertion of the DvCRP1 gene into yeast for use in bio-refineries during fermentation of heavy metals-contaminated substrates. In addition, this is a promising method for phytoremediation of agricultural soils highly contaminated by heavy metals.

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

PubMed Central

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

Production scheduling with discrete and renewable additional resources

NASA Astrophysics Data System (ADS)

Kalinowski, K.; Grabowik, C.; Paprocka, I.; Kempa, W.

2015-11-01

In this paper an approach to planning of additional resources when scheduling operations are discussed. The considered resources are assumed to be discrete and renewable. In most research in scheduling domain, the basic and often the only type of regarded resources is a workstation. It can be understood as a machine, a device or even as a separated space on the shop floor. In many cases, during the detailed scheduling of operations the need of using more than one resource, required for its implementation, can be indicated. Resource requirements for an operation may relate to different resources or resources of the same type. Additional resources are most often referred to these human resources, tools or equipment, for which the limited availability in the manufacturing system may have an influence on the execution dates of some operations. In the paper the concept of the division into basic and additional resources and their planning method was shown. A situation in which sets of basic and additional resources are not separable - the same additional resource may be a basic resource for another operation is also considered. Scheduling of operations, including greater amount of resources can cause many difficulties, depending on whether the resource is involved in the entire time of operation, only in the selected part(s) of operation (e.g. as auxiliary staff at setup time) or cyclic - e.g. when an operator supports more than one machine, or supervises the execution of several operations. For this reason the dates and work times of resources participation in the operation can be different. Presented issues are crucial when modelling of production scheduling environment and designing of structures for the purpose of scheduling software development.

Safely coupling livestock and crop production systems: how rapidly do antibiotic resistance genes dissipate in soil following a commercial application of swine or dairy manure?

PubMed

Marti, Romain; Tien, Yuan-Ching; Murray, Roger; Scott, Andrew; Sabourin, Lyne; Topp, Edward

2014-05-01

Animal manures recycled onto crop production land carry antibiotic-resistant bacteria. The present study evaluated the fate in soil of selected genes associated with antibiotic resistance or genetic mobility in field plots cropped to vegetables and managed according to normal farming practice. Referenced to unmanured soil, fertilization with swine or dairy manure increased the relative abundance of the gene targets sul1, erm(B), str(B), int1, and IncW repA. Following manure application in the spring of 2012, gene copy number decayed exponentially, reaching background levels by the fall of 2012. In contrast, gene copy number following manure application in the fall of 2012 or spring of 2013 increased significantly in the weeks following application and then declined. In both cases, the relative abundance of gene copy numbers had not returned to background levels by the fall of 2013. Overall, these results suggest that under conditions characteristic of agriculture in a humid continental climate, a 1-year period following a commercial application of raw manure is sufficient to ensure that an additional soil burden of antibiotic resistance genes approaches background. The relative abundance of several gene targets exceeded background during the growing season following a spring application or an application done the previous fall. Results from the present study reinforce the advisability of treating manure prior to use in crop production systems.

The use of NaCl addition for the improvement of polyhydroxyalkanoate production by Cupriavidus necator.

PubMed

Passanha, Pearl; Kedia, Gopal; Dinsdale, Richard M; Guwy, Alan J; Esteves, Sandra R

2014-07-01

External stress factors in the form of ionic species or temperature increases have been shown to produce a stress response leading to enhanced PHA production. The effect of five different NaCl concentrations, namely 3.5, 6.5, 9, 12 and 15 g/l NaCl on PHA productivity using Cupriavidus necator has been investigated alongside a control (no added NaCl). A dielectric spectroscopy probe was used to measure PHA accumulation online in conjunction with the chemical offline analysis of PHA. The highest PHA production was obtained with the addition of 9 g/l NaCl, which yielded 30% higher PHA than the control. Increasing the addition of NaCl to 15 g/l was found to inhibit the production of PHA. NaCl addition can therefore be used as a simple, low cost, sustainable, non toxic and non reactive external stress strategy for increasing PHA productivity. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kokusho, Ryuhei, E-mail: kokusho@ss.ab.a.u-tokyo.a

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 regulationmore » 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.« less

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

PubMed

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.

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

NASA Astrophysics Data System (ADS)

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.

Sparse Additive Ordinary Differential Equations for Dynamic Gene Regulatory Network Modeling.

PubMed

Wu, Hulin; Lu, Tao; Xue, Hongqi; Liang, Hua

2014-04-02

The gene regulation network (GRN) is a high-dimensional complex system, which can be represented by various mathematical or statistical models. The ordinary differential equation (ODE) model is one of the popular dynamic GRN models. High-dimensional linear ODE models have been proposed to identify GRNs, but with a limitation of the linear regulation effect assumption. In this article, we propose a sparse additive ODE (SA-ODE) model, coupled with ODE estimation methods and adaptive group LASSO techniques, to model dynamic GRNs that could flexibly deal with nonlinear regulation effects. The asymptotic properties of the proposed method are established and simulation studies are performed to validate the proposed approach. An application example for identifying the nonlinear dynamic GRN of T-cell activation is used to illustrate the usefulness of the proposed method.

Production of Low-Freezing-Point Highly Branched Alkanes through Michael Addition.

PubMed

Jing, Yaxuan; Xia, Qineng; Liu, Xiaohui; Wang, Yanqin

2017-12-22

A new approach for the production of low-freezing-point, high-quality fuels from lignocellulose-derived molecules was developed with Michael addition as the key step. Among the investigated catalysts, CoCl 2 ⋅6 H 2 O was found most active for the Michael addition of 2,4-pentanedione with FA (single aldol adduct of furfural and acetone, 4-(2-furanyl)-3-butene-2-one). Over CoCl 2 ⋅6 H 2 O, a high carbon yield of C 13 oxygenates (about 75 %) can be achieved under mild conditions (353 K, 20 h). After hydrodeoxygenation, low-freezing-point (<223 K) branched alkanes with 13 carbons within jet fuel ranges were obtained over a Pd/NbOPO 4 catalyst. Furthermore, C 18,23 fuel precursors could be easily synthesized through Michael addition of 2,4-pentanedione with DFA (double-condensation product of furfural and acetone) under mild conditions and the molar ratio of C 18 /C 23 is dependent on the reaction conditions of Michael addition. After hydrodeoxygenation, high density (0.8415 g mL -1 ) and low-freezing-point (<223 K) branched alkanes with 18, 23 carbons within lubricant range were also obtained over a Pd/NbOPO 4 catalyst. These highly branched alkanes can be directly used as transportation fuels or additives. This work opens a new strategy for the synthesis of highly branched alkanes with low freezing point from renewable biomass. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Toxicological safety assessment of genetically modified Bacillus thuringiensis with additional N-acyl homoserine lactonase gene.

PubMed

Peng, Donghai; Zhou, Chenfei; Chen, Shouwen; Ruan, Lifang; Yu, Ziniu; Sun, Ming

2008-01-01

The aim of the present study is to evaluate the toxicology safety to mammals of a genetically modified (GM) Bacillus thuringiensis with an additional N-acyl homoserine lactones gene (aiiA), which possesses insecticidal activity together with restraint of bacterial pathogenicity and is intended for use as a multifunctional biopesticide. Safety assessments included an acute oral toxicity test and 28-d animal feeding study in Wistar rats, primary eye and dermal irritation in Zealand White rabbits, and delayed contact hypersensitivity in guinea pigs. Tests were conducted using spray-dried powder preparation. This GM product showed toxicity neither in oral acute toxicity test nor in 28-d animal feeding test at a dose of 5,000 mg/kg body weight. During the animal feeding test, there were no significant differences in growth, food and water consumption, hematology, blood biochemical indices, organ weights, and histopathology finding between rats in controls and tested groups. Tested animals in primary eye and dermal irritation and delayed contact hypersensitivity test were also devoid of any toxicity compared to controls. All the above results demonstrated that the GM based multifunctional B. thuringiensis has low toxicity and low eye and dermal irritation and would not cause hypersensitivity to laboratory mammals and therefore could be regarded as safe for use as a pesticide.

[Effects of menthol as an additive in tobacco products and the need for regulation].

PubMed

Kahnert, S; Nair, U; Mons, U; Pötschke-Langer, M

2012-03-01

Menthol is the most widely used and the most prominent tobacco additive in tobacco products advertised and marketed by the tobacco industry. Besides its characteristic flavor, it possesses a variety of pharmacological properties facilitating tobacco smoke inhalation and potentiating dependence. These properties of menthol not only favor tobacco initiation and consumption but can also prevent smoking cessation. This article summarizes the effect of menthol as an additive in tobacco products and its effect on tobacco consumption that causes a number of chronic diseases and premature death and, therefore, counteracts tobacco control measures. Currently, there is no legislative regulation in Germany that considers the health hazard, addiction-enhancing and attractiveness-increasing properties of additives permitted in tobacco products. Effective regulation or even a ban could contribute to a reduction of tobacco consumption and, hence, save many people from a long-lasting tobacco dependence.

Inorganic carbon addition stimulates snow algae primary productivity

NASA Astrophysics Data System (ADS)

Hamilton, T. L.; Havig, J. R.

2017-12-01

Earth has experienced glacial/interglacial oscillations throughout its history. Today over 15 million square kilometers (5.8 million square miles) of Earth's land surface is covered in ice including glaciers, ice caps, and the ice sheets of Greenland and Antarctica, most of which are retreating as a consequence of increased atmospheric CO2. Glaciers are teeming with life and supraglacial snow and ice surfaces are often red due to blooms of photoautotrophic algae. Recent evidence suggests the red pigmentation, secondary carotenoids produced in part to thrive under high irradiation, lowers albedo and accelerates melt. However, there are relatively few studies that report the productivity of snow algae communities and the parameters that constrain their growth on snow and ice surfaces. Here, we demonstrate that snow algae primary productivity can be stimulated by the addition of inorganic carbon. We found an increase in light-dependent carbon assimilation in snow algae microcosms amended with increasing amounts of inorganic carbon. Our snow algae communities were dominated by typical cosmopolitan snow algae species recovered from Alpine and Arctic environments. The climate feedbacks necessary to enter and exit glacial/interglacial oscillations are poorly understood. Evidence and models agree that global Snowball events are accompanied by changes in atmospheric CO2 with increasing CO2 necessary for entering periods of interglacial time. Our results demonstrate a positive feedback between increased CO2 and snow algal productivity and presumably growth. With the recent call for bio-albedo effects to be considered in climate models, our results underscore the need for robust climate models to include feedbacks between supraglacial primary productivity, albedo, and atmospheric CO2.

Overexpressing enzymes of the Ehrlich pathway and deleting genes of the competing pathway in Saccharomyces cerevisiae for increasing 2-phenylethanol production from glucose.

PubMed

Shen, Li; Nishimura, Yuya; Matsuda, Fumio; Ishii, Jun; Kondo, Akihiko

2016-07-01

2-Phenylethanol (2-PE) is a higher aromatic alcohol that is used in the cosmetics and food industries. The budding yeast Saccharomyces cerevisiae is considered to be a suitable host for the industrial production of higher alcohols, including 2-PE. To produce 2-PE from glucose in S. cerevisiae, we searched for suitable 2-keto acid decarboxylase (KDC) and alcohol dehydrogenase (ADH) enzymes of the Ehrlich pathway for overexpression in strain YPH499, and found that overexpression of the ARO10 and/or ADH1 genes increased 2-PE production from glucose. Further, we screened ten BY4741 single-deletion mutants of genes involved in the competing pathways for 2-PE production, and found that strains aro8Δ and aat2Δ displayed increased 2-PE production. Based on these results, we engineered a BY4741 strain that overexpressed ARO10 and contained an aro8Δ deletion, and demonstrated that the strain produced 96 mg/L 2-PE from glucose as the sole carbon source. As this engineered S. cerevisiae strain showed a significant increase in 2-PE production from glucose without the addition of an intermediate carbon substrate, it is a promising candidate for the large-scale production of 2-PE. Copyright © 2016. Published by Elsevier B.V.

Systems Pharmacology-Based Discovery of Natural Products for Precision Oncology Through Targeting Cancer Mutated Genes.

PubMed

Fang, J; Cai, C; Wang, Q; Lin, P; Zhao, Z; Cheng, F

2017-03-01

Massive cancer genomics data have facilitated the rapid revolution of a novel oncology drug discovery paradigm through targeting clinically relevant driver genes or mutations for the development of precision oncology. Natural products with polypharmacological profiles have been demonstrated as promising agents for the development of novel cancer therapies. In this study, we developed an integrated systems pharmacology framework that facilitated identifying potential natural products that target mutated genes across 15 cancer types or subtypes in the realm of precision medicine. High performance was achieved for our systems pharmacology framework. In case studies, we computationally identified novel anticancer indications for several US Food and Drug Administration-approved or clinically investigational natural products (e.g., resveratrol, quercetin, genistein, and fisetin) through targeting significantly mutated genes in multiple cancer types. In summary, this study provides a powerful tool for the development of molecularly targeted cancer therapies through targeting the clinically actionable alterations by exploiting the systems pharmacology of natural products. © 2017 The Authors CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals, Inc. on behalf of American Society for Clinical Pharmacology and Therapeutics.

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

PubMed

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.

Identification of a Lytic-Cycle Epstein-Barr Virus Gene Product That Can Regulate PKR Activation

PubMed Central

Poppers, Jeremy; Mulvey, Matthew; Perez, Cesar; Khoo, David; Mohr, Ian

2003-01-01

The Epstein-Barr virus (EBV) SM protein is a posttranscriptional regulator of viral gene expression. Like many transactivators encoded by herpesviruses, SM transports predominantly unspliced viral mRNA cargo from the nucleus to the cytosol, where it is subsequently translated. This activity likely involves a region of the protein that has homology to the herpes simplex virus type 1 (HSV-1) ICP27 gene product, the first member of this class of regulators to be discovered. However, SM also contains a repetitive segment rich in arginine and proline residues that is dispensable for its effects on RNA transport and splicing. This portion of SM, comprised of RXP triplet repeats, shows homology to the carboxyl-terminal domain of Us11, a double-stranded RNA (dsRNA) binding protein encoded by HSV-1 that inhibits activation of the cellular PKR kinase. To evaluate the intrinsic ability of SM to regulate PKR, we expressed and purified several SM protein derivatives and examined their activity in a variety of biochemical assays. The full-length SM protein bound dsRNA, associated physically with PKR, and prevented PKR activation. Removal of the 37-residue RXP domain significantly compromised all of these activities. Furthermore, the SM RXP domain was itself sufficient to inhibit PKR activation and interact with the kinase. Relative to its Us11 counterpart, the SM RXP segment bound dsRNA with reduced affinity and responded differently to single-stranded competitor polynucleotides. Thus, SM represents the first EBV gene product expressed during the lytic cycle that can prevent PKR activation. In addition, the RXP repeat segment appears to be a conserved herpesvirus motif capable of associating with dsRNA and modulating activation of the PKR kinase, a molecule important for the control of translation and the cellular antiviral response. PMID:12477828

Identification of a lytic-cycle Epstein-Barr virus gene product that can regulate PKR activation.

PubMed

Poppers, Jeremy; Mulvey, Matthew; Perez, Cesar; Khoo, David; Mohr, Ian

2003-01-01

The Epstein-Barr virus (EBV) SM protein is a posttranscriptional regulator of viral gene expression. Like many transactivators encoded by herpesviruses, SM transports predominantly unspliced viral mRNA cargo from the nucleus to the cytosol, where it is subsequently translated. This activity likely involves a region of the protein that has homology to the herpes simplex virus type 1 (HSV-1) ICP27 gene product, the first member of this class of regulators to be discovered. However, SM also contains a repetitive segment rich in arginine and proline residues that is dispensable for its effects on RNA transport and splicing. This portion of SM, comprised of RXP triplet repeats, shows homology to the carboxyl-terminal domain of Us11, a double-stranded RNA (dsRNA) binding protein encoded by HSV-1 that inhibits activation of the cellular PKR kinase. To evaluate the intrinsic ability of SM to regulate PKR, we expressed and purified several SM protein derivatives and examined their activity in a variety of biochemical assays. The full-length SM protein bound dsRNA, associated physically with PKR, and prevented PKR activation. Removal of the 37-residue RXP domain significantly compromised all of these activities. Furthermore, the SM RXP domain was itself sufficient to inhibit PKR activation and interact with the kinase. Relative to its Us11 counterpart, the SM RXP segment bound dsRNA with reduced affinity and responded differently to single-stranded competitor polynucleotides. Thus, SM represents the first EBV gene product expressed during the lytic cycle that can prevent PKR activation. In addition, the RXP repeat segment appears to be a conserved herpesvirus motif capable of associating with dsRNA and modulating activation of the PKR kinase, a molecule important for the control of translation and the cellular antiviral response.

An ergot alkaloid biosynthesis gene and clustered hypothetical genes from Aspergillus fumigatus.

PubMed

Coyle, Christine M; Panaccione, Daniel G

2005-06-01

The ergot alkaloids are a family of indole-derived mycotoxins with a variety of significant biological activities. Aspergillus fumigatus, a common airborne fungus and opportunistic human pathogen, and several fungi in the relatively distant taxon Clavicipitaceae (clavicipitaceous fungi) produce different sets of ergot alkaloids. The ergot alkaloids of these divergent fungi share a four-member ergoline ring but differ in the number, type, and position of the side chains. Several genes required for ergot alkaloid production are known in the clavicipitaceous fungi, and these genes are clustered in the genome of the ergot fungus Claviceps purpurea. We investigated whether the ergot alkaloids of A. fumigatus have a common biosynthetic and genetic origin with those of the clavicipitaceous fungi. A homolog of dmaW, the gene controlling the determinant step in the ergot alkaloid pathway of clavicipitaceous fungi, was identified in the A. fumigatus genome. Knockout of dmaW eliminated all known ergot alkaloids from A. fumigatus, and complementation of the mutation restored ergot alkaloid production. Clustered with dmaW in the A. fumigatus genome are sequences corresponding to five genes previously proposed to encode steps in the ergot alkaloid pathway of C. purpurea, as well as additional sequences whose deduced protein products are consistent with their involvement in the ergot alkaloid pathway. The corresponding genes have similarities in their nucleotide sequences, but the orientations and positions within the cluster of several of these genes differ. The data indicate that the ergot alkaloid biosynthetic capabilities in A. fumigatus and the clavicipitaceous fungi had a common origin.

An Ergot Alkaloid Biosynthesis Gene and Clustered Hypothetical Genes from Aspergillus fumigatus†

PubMed Central

Coyle, Christine M.; Panaccione, Daniel G.

2005-01-01

The ergot alkaloids are a family of indole-derived mycotoxins with a variety of significant biological activities. Aspergillus fumigatus, a common airborne fungus and opportunistic human pathogen, and several fungi in the relatively distant taxon Clavicipitaceae (clavicipitaceous fungi) produce different sets of ergot alkaloids. The ergot alkaloids of these divergent fungi share a four-member ergoline ring but differ in the number, type, and position of the side chains. Several genes required for ergot alkaloid production are known in the clavicipitaceous fungi, and these genes are clustered in the genome of the ergot fungus Claviceps purpurea. We investigated whether the ergot alkaloids of A. fumigatus have a common biosynthetic and genetic origin with those of the clavicipitaceous fungi. A homolog of dmaW, the gene controlling the determinant step in the ergot alkaloid pathway of clavicipitaceous fungi, was identified in the A. fumigatus genome. Knockout of dmaW eliminated all known ergot alkaloids from A. fumigatus, and complementation of the mutation restored ergot alkaloid production. Clustered with dmaW in the A. fumigatus genome are sequences corresponding to five genes previously proposed to encode steps in the ergot alkaloid pathway of C. purpurea, as well as additional sequences whose deduced protein products are consistent with their involvement in the ergot alkaloid pathway. The corresponding genes have similarities in their nucleotide sequences, but the orientations and positions within the cluster of several of these genes differ. The data indicate that the ergot alkaloid biosynthetic capabilities in A. fumigatus and the clavicipitaceous fungi had a common origin. PMID:15933009

Diversity of Antibiotic Resistance Genes in Enterococcus Strains Isolated from Ready-to-Eat Meat Products.

PubMed

Chajęcka-Wierzchowska, Wioleta; Zadernowska, Anna; Łaniewska-Trokenheim, Łucja

2016-10-25

The objective of the study was to answer the question of whether the ready-to-eat meat products can pose indirect hazard for consumer health serving as reservoir of Enterococcus strains harboring tetracyclines, aminoglycosides, and macrolides resistance genes. A total of 390 samples of ready-to-eat meat products were investigated. Enterococcus strains were found in 74.1% of the samples. A total of 302 strains were classified as: Enterococcus faecalis (48.7%), Enterococcus faecium (39.7%), Enterococcus casseliflavus (4.3%), Enterococcus durans (3.0%), Enterococcus hirae (2.6%), and other Enterococcus spp. (1.7%). A high percentage of isolates were resistant to streptomycin high level (45%) followed by erythromycin (42.7%), fosfomycin (27.2%), rifampicin (19.2%), tetracycline (36.4%), tigecycline (19.9%). The ant(6')-Ia gene was the most frequently found gene (79.6%). Among the other genes that encode aminoglycosides-modifying enzymes, the highest portion of the strains had the aac(6')-Ie-aph(2'')-Ia (18.5%) and aph(3'')-IIIa (16.6%), but resistance of isolates from food is also an effect of the presence of aph(2'')-Ib, aph(2'')-Ic, aph(2'')-Id genes. Resistance to tetracyclines was associated with the presence of tetM (43.7%), tetL (32.1%), tetK (14.6%), tetW (0.7%), and tetO (0.3%) genes. The ermB and ermA genes were found in 33.8% and 18.9% of isolates, respectively. Nearly half of the isolates contained a conjugative transposon of the Tn916/Tn1545 family. Enterococci are widely present in retail ready-to-eat meat products. Many isolated strains (including such species as E. casseliflavus, E. durans, E. hirae, and Enterococcus gallinarum) are antibiotic resistant and carry transferable resistance genes. © 2016 Institute of Food Technologists®.

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

PubMed

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.

Methods to identify and analyze gene products involved in neuronal intracellular transport using Drosophila

PubMed Central

Neisch, Amanda L.; Avery, Adam W.; Machame, James B.; Li, Min-gang; Hays, Thomas S.

2017-01-01

Proper neuronal function critically depends on efficient intracellular transport and disruption of transport leads to neurodegeneration. Molecular pathways that support or regulate neuronal transport are not fully understood. A greater understanding of these pathways will help reveal the pathological mechanisms underlying disease. Drosophila melanogaster is the premier model system for performing large-scale genetic functional screens. Here we describe methods to carry out primary and secondary genetic screens in Drosophila aimed at identifying novel gene products and pathways that impact neuronal intracellular transport. These screens are performed using whole animal or live cell imaging of intact neural tissue to ensure integrity of neurons and their cellular environment. The primary screen is used to identify gross defects in neuronal function indicative of a disruption in microtubule-based transport. The secondary screens, conducted in both motoneurons and dendritic arborization neurons, will confirm the function of candidate gene products in intracellular transport. Together, the methodologies described here will support labs interested in identifying and characterizing gene products that alter intracellular transport in Drosophila. PMID:26794520

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

PubMed

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.

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

PubMed

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

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

PubMed Central

Oberding, Lisa K.

2017-01-01

ABSTRACT Paraffinic n-alkanes (>C17) 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 (C28H58). 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 C28H58 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 C28H58-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 C28H58-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 applications for

Roles of the 2 microns gene products in stable maintenance of the 2 microns plasmid of Saccharomyces cerevisiae.

PubMed Central

Reynolds, A E; Murray, A W; Szostak, J W

1987-01-01

We have examined the replication and segregation of the Saccharomyces cerevisiae 2 microns circle. The amplification of the plasmid at low copy numbers requires site-specific recombination between the 2 microns inverted repeat sequences catalyzed by the plasmid-encoded FLP gene. No other 2 microns gene products are required. The overexpression of FLP in a strain carrying endogenous 2 microns leads to uncontrolled plasmid replication, longer cell cycles, and cell death. Two different assays show that the level of Flp activity decreases with increasing 2 microns copy number. This regulation requires the products of the REP1 and REP2 genes. These gene products also act together to ensure that 2 microns molecules are randomly segregated between mother and daughter cells at cell division. Images PMID:3316982

CO2 Reduction Selective for C≥2 Products on Polycrystalline Copper with N-Substituted Pyridinium Additives.

PubMed

Han, Zhiji; Kortlever, Ruud; Chen, Hsiang-Yun; Peters, Jonas C; Agapie, Theodor

2017-08-23

Electrocatalytic CO 2 reduction to generate multicarbon products is of interest for applications in artificial photosynthetic schemes. This is a particularly attractive goal for CO 2 reduction by copper electrodes, where a broad range of hydrocarbon products can be generated but where selectivity for C-C coupled products relative to CH 4 and H 2 remains an impediment. Herein we report a simple yet highly selective catalytic system for CO 2 reduction to C ≥2 hydrocarbons on a polycrystalline Cu electrode in bicarbonate aqueous solution that uses N-substituted pyridinium additives. Selectivities of 70-80% for C 2 and C 3 products with a hydrocarbon ratio of C ≥2 /CH 4 significantly greater than 100 have been observed with several additives. 13 C-labeling studies verify CO 2 to be the sole carbon source in the C ≥2 hydrocarbons produced. Upon electroreduction, the N-substituted pyridinium additives lead to film deposition on the Cu electrode, identified in one case as the reductive coupling product of N -arylpyridinium. Product selectivity can also be tuned from C ≥2 species to H 2 (∼90%) while suppressing methane with certain N-heterocyclic additives.

The dam replacing gene product enhances Neisseria gonorrhoeae FA1090 viability and biofilm formation

PubMed Central

Kwiatek, Agnieszka; Bacal, Pawel; Wasiluk, Adrian; Trybunko, Anastasiya; Adamczyk-Poplawska, Monika

2014-01-01

Many Neisseriaceae do not exhibit Dam methyltransferase activity and, instead of the dam gene, possess drg (dam replacing gene) inserted in the leuS/dam locus. The drg locus in Neisseria gonorrhoeae FA1090 has a lower GC-pairs content (40.5%) compared to the whole genome of N. gonorrhoeae FA1090 (52%). The gonococcal drg gene encodes a DNA endonuclease Drg, with GmeATC specificity. Disruption of drg or insertion of the dam gene in gonococcal genome changes the level of expression of genes as shown by transcriptome analysis. For the drg-deficient N. gonorrhoeae mutant, a total of 195 (8.94% of the total gene pool) genes exhibited an altered expression compared to the wt strain by at least 1.5 fold. In dam-expressing N. gonorrhoeae mutant, the expression of 240 genes (11% of total genes) was deregulated. Most of these deregulated genes were involved in translation, DNA repair, membrane biogenesis and energy production as shown by cluster of orthologous group analysis. In vivo, the inactivation of drg gene causes the decrease of the number of live neisserial cells and long lag phase of growth. The insertion of dam gene instead of drg locus restores cell viability. We have also shown that presence of the drg gene product is important for N. gonorrhoeae FA1090 in adhesion, including human epithelial cells, and biofilm formation. Biofilm produced by drg-deficient strain is formed by more dispersed cells, compared to this one formed by parental strain as shown by scanning electron and confocal microscopy. Also adherence assays show a significantly smaller biomass of formed biofilm (OD570 = 0.242 ± 0.038) for drg-deficient strain, compared to wild-type strain (OD570 = 0.378 ± 0.057). Dam-expressing gonococcal cells produce slightly weaker biofilm with cells embedded in an extracellular matrix. This strain has also a five times reduced ability for adhesion to human epithelial cells. In this context, the presence of Drg is more advantageous for N. gonorrhoeae biology than

TAC1 Gene Products Regulate Pituitary Hormone Secretion and Gene Expression in Prepubertal Grass Carp Pituitary Cells.

PubMed

Hu, Guangfu; He, Mulan; Ko, Wendy K W; Wong, Anderson O L

2017-06-01

Tachykinin-1 (TAC1) is known to have diverse functions in mammals, but similar information is scarce in fish species. Using grass carp as a model, the pituitary actions, receptor specificity and postreceptor signaling of TAC1 gene products, namely substance P (SP) and neurokinin A (NKA), were examined. TAC1 encoding SP and NKA as well as tachykinin receptors NK1R and NK2R were cloned in the carp pituitary. The newly cloned receptors were shown to be functional with properties similar to mammalian counterparts. In carp pituitary cells, SP and NKA could trigger luteinizing hormone (LH), prolactin (PRL), and somatolactin α (SLα) secretion, with parallel rises in PRL and SLα transcripts. Short-term SP treatment (3 hours) induced LH release, whereas prolonged induction (24 hours) could attenuate LHβ messenger RNA (mRNA) expression. At pituitary cell level, LH, PRL, and SLα regulation by TAC1 gene products were mediated by NK1R, NK2R, and NK3R, respectively. Apparently, SP- and NKA-induced LH and SLα secretion and transcript expression were mediated by adenylyl cyclase/cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA), phospholiphase C (PLC)/inositol 1,4,5-triphosphate/protein kinase C (PKC), and Ca2+/calmodulin (CaM)/CaM-dependent protein kinase-II pathways. The signal transduction for PRL responses was similar, except for the absence of a PKC component. Regarding SP inhibition of LHβ mRNA expression, the cAMP/PKA- and PLC/PKC-dependent (but not Ca2+/CaM-dependent) cascades were involved. These results, as a whole, suggest that TAC1 gene products play a role in LH, PRL, and SLα regulation via overlapping postreceptor signaling coupled to different subtypes of tachykinin receptor expressed in the carp pituitary. Copyright © 2017 Endocrine Society.

The BnGRF2 gene (GRF2-like gene from Brassica napus) enhances seed oil production through regulating cell number and plant photosynthesis

PubMed Central

Liu, Jing; Hua, Wei; Yang, Hong-Li; Zhan, Gao-Miao; Deng, Lin-Bin; Wang, Xin-Fa; Liu, Gui-Hua; Wang, Han-Zhong

2012-01-01

Seed yield and oil content are two important agricultural characteristics in oil crop breeding, and a lot of functional gene research is being concentrated on increasing these factors. In this study, by differential gene expression analyses between rapeseed lines (zy036 and 51070) which exhibit different levels of seed oil production, BnGRF2 (Brassica napus growth-regulating factor 2-like gene) was identified in the high oil-producing line zy036. To elucidate the possible roles of BnGRF2 in seed oil production, the cDNA sequences of the rapeseed GRF2 gene were isolated. The Blastn result showed that rapeseed contained BnGRF2a/2b which were located in the A genome (A1 and A3) and C genome (C1 and C6), respectively, and the dominantly expressed gene BnGRF2a was chosen for transgenic research. Analysis of 35S-BnGRF2a transgenic Arabidopsis showed that overexpressed BnGRF2a resulted in an increase in seed oil production of >50%. Moreover, BnGRF2a also induced a >20% enlargement in extended leaves and >40% improvement in photosynthetic efficiency because of an increase in the chlorophyll content. Furthermore, transcriptome analyses indicated that some genes associated with cell proliferation, photosynthesis, and oil synthesis were up-regulated, which revealed that cell number and plant photosynthesis contributed to the increased seed weight and oil content. Because of less efficient self-fertilization induced by the longer pistil in the 35S-BnGRF2a transgenic line, Napin-BnGRF2a transgenic lines were further used to identify the function of BnGRF2, and the results showed that seed oil production also could increase >40% compared with the wild-type control. The results suggest that improvement to economically important characteristics in oil crops may be achieved by manipulation of the GRF2 expression level. PMID:22442419

The BnGRF2 gene (GRF2-like gene from Brassica napus) enhances seed oil production through regulating cell number and plant photosynthesis.

PubMed

Liu, Jing; Hua, Wei; Yang, Hong-Li; Zhan, Gao-Miao; Li, Rong-Jun; Deng, Lin-Bin; Wang, Xin-Fa; Liu, Gui-Hua; Wang, Han-Zhong

2012-06-01

Seed yield and oil content are two important agricultural characteristics in oil crop breeding, and a lot of functional gene research is being concentrated on increasing these factors. In this study, by differential gene expression analyses between rapeseed lines (zy036 and 51070) which exhibit different levels of seed oil production, BnGRF2 (Brassica napus growth-regulating factor 2-like gene) was identified in the high oil-producing line zy036. To elucidate the possible roles of BnGRF2 in seed oil production, the cDNA sequences of the rapeseed GRF2 gene were isolated. The Blastn result showed that rapeseed contained BnGRF2a/2b which were located in the A genome (A1 and A3) and C genome (C1 and C6), respectively, and the dominantly expressed gene BnGRF2a was chosen for transgenic research. Analysis of 35S-BnGRF2a transgenic Arabidopsis showed that overexpressed BnGRF2a resulted in an increase in seed oil production of >50%. Moreover, BnGRF2a also induced a >20% enlargement in extended leaves and >40% improvement in photosynthetic efficiency because of an increase in the chlorophyll content. Furthermore, transcriptome analyses indicated that some genes associated with cell proliferation, photosynthesis, and oil synthesis were up-regulated, which revealed that cell number and plant photosynthesis contributed to the increased seed weight and oil content. Because of less efficient self-fertilization induced by the longer pistil in the 35S-BnGRF2a transgenic line, Napin-BnGRF2a transgenic lines were further used to identify the function of BnGRF2, and the results showed that seed oil production also could increase >40% compared with the wild-type control. The results suggest that improvement to economically important characteristics in oil crops may be achieved by manipulation of the GRF2 expression level.

Application of an Efficient Gene Targeting System Linking Secondary Metabolites to their Biosynthetic Genes in Aspergillus terreus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guo, Chun-Jun; Knox, Benjamin P.; Sanchez, James F.

2013-07-19

Nonribosomal peptides (NRPs) are natural products biosynthesized by NRP synthetases. A kusA-, pyrG- mutant strain of Aspergillusterreus NIH 2624 was developed that greatly facilitated the gene targeting efficiency in this organism. Application of this tool allowed us to link four major types of NRP related secondary metabolites to their responsible genes in A. terreus. In addition, an NRP related melanin synthetase was also identified in this species.

Bacterial avirulence genes.

PubMed

Leach, J E; White, F F

1996-01-01

Although more than 30 bacterial avirulence genes have been cloned and characterized, the function of the gene products in the elictitation of resistance is unknown in all cases but one. The product of avrD from Pseudomonas syringae pv. glycinea likely functions indirectly to elicit resistance in soybean, that is, evidence suggests the gene product is an enzyme involved in elicitor production. In most if not all cases, bacterial avirulence gene function is dependent on interactions with the hypersensitive response and pathogenicity (hrp) genes. Many hrp genes are similar to genes involved in delivery of pathogenicity factors in mammalian bacterial pathogens. Thus, analogies between mammalian and plant pathogens may provide needed clues to elucidate how virulence gene products control induction of resistance.

Cloning and Characterization of GLOSSY1, a Maize Gene Involved in Cuticle Membrane and Wax Production1[w

PubMed Central

Sturaro, Monica; Hartings, Hans; Schmelzer, Elmon; Velasco, Riccardo; Salamini, Francesco; Motto, Mario

2005-01-01

The cuticle covering the aerial organs of land plants plays a protective role against several biotic and abiotic stresses and, in addition, participates in a variety of plant-insect interactions. Here, we describe the molecular cloning and characterization of the maize (Zea mays) GLOSSY1 (GL1) gene, a component of the pathway leading to cuticular wax biosynthesis in seedling leaves. The genomic and cDNA sequences we isolated differ significantly in length and in most of the coding region from those previously identified. The predicted GL1 protein includes three histidine-rich domains, the landmark of a family of membrane-bound desaturases/hydroxylases, including fatty acid-modifying enzymes. GL1 expression is not restricted to the juvenile developmental stage of the maize plant, pointing to a broader function of the gene product than anticipated on the basis of the mutant phenotype. Indeed, in addition to affecting cuticular wax biosynthesis, gl1 mutations have a pleiotropic effect on epidermis development, altering trichome size and impairing cutin structure. Of the many wax biosynthetic genes identified so far, only a few from Arabidopsis (Arabidopsis thaliana) were found to be essential for normal cutin formation. Among these is WAX2, which shares 62% identity with GL1 at the protein level. In wax2-defective plants, cutin alterations induce postgenital organ fusion. This trait is not displayed by gl1 mutants, suggesting a different role of the maize and Arabidopsis cuticle in plant development. PMID:15849306

Characterization of the Drosophila Methoprene -tolerant gene product. Juvenile hormone binding and ligand-dependent gene regulation.

PubMed

Miura, Ken; Oda, Masahito; Makita, Sumiko; Chinzei, Yasuo

2005-03-01

Juvenile hormones (JHs) of insects are sesquiterpenoids that regulate a great diversity of processes in development and reproduction. As yet the molecular modes of action of JH are poorly understood. The Methoprene-tolerant (Met) gene of Drosophila melanogaster has been found to be responsible for resistance to a JH analogue (JHA) insecticide, methoprene. Previous studies on Met have implicated its involvement in JH signaling, although direct evidence is lacking. We have now examined the product of Met (MET) in terms of its binding to JH and ligand-dependent gene regulation. In vitro synthesized MET directly bound to JH III with high affinity (Kd = 5.3 +/- 1.5 nm, mean +/- SD), consistent with the physiological JH concentration. In transient transfection assays using Drosophila S2 cells the yeast GAL4-DNA binding domain fused to MET exerted JH- or JHA-dependent activation of a reporter gene. Activation of the reporter gene was highly JH- or JHA-specific with the order of effectiveness: JH III > JH II > JH I > methoprene; compounds which are only structurally related to JH or JHA did not induce any activation. Localization of MET in the S2 cells was nuclear irrespective of the presence or absence of JH. These results suggest that MET may function as a JH-dependent transcription factor.

30 CFR 250.1629 - Additional production and fuel gas system requirements.

Code of Federal Regulations, 2010 CFR

2010-07-01

.... (4) Fire- and gas-detection system. (i) Fire (flame, heat, or smoke) sensors shall be installed in all enclosed classified areas. Gas sensors shall be installed in all inadequately ventilated, enclosed... 30 Mineral Resources 2 2010-07-01 2010-07-01 false Additional production and fuel gas system...

30 CFR 250.1629 - Additional production and fuel gas system requirements.

Code of Federal Regulations, 2011 CFR

2011-07-01

... structure. (4) Fire- and gas-detection system. (i) Fire (flame, heat, or smoke) sensors shall be installed in all enclosed classified areas. Gas sensors shall be installed in all inadequately ventilated... 30 Mineral Resources 2 2011-07-01 2011-07-01 false Additional production and fuel gas system...

Social aspects in additive manufacturing of pharmaceutical products.

PubMed

Lind, Johanna; Kälvemark Sporrong, Sofia; Kaae, Susanne; Rantanen, Jukka; Genina, Natalja

2017-08-01

Additive manufacturing (AM) techniques, such as drug printing, represent a new engineering approach that can implement the concept of personalized medicine via on-demand manufacturing of dosage forms with individually adjusted doses. Implementation of AM principles, such as pharmacoprinting, will challenge the entire drug distribution chain and affect the society at different levels. Areas covered: This work summarizes the concept of personalized medicine and gives an overview of possibilities for monitoring patients' health. The most recent activities in the field of printing technologies for fabrication of dosage forms and 'polypills' with flexible doses and tailored release profiles are reviewed. Different scenarios for the drug distribution chain with the required adjustments in drug logistics, quality systems and environmental safety are discussed, as well as whether AM will be used for production of on-demand medicine. The impact of such changes in the distribution chain on regulation, healthcare professionals and patients are highlighted. Expert opinion: Drug manufacturing by traditional methods is well-established, but it lacks the possibility for on-demand personalized drug production. With the recent approval of the first printed medicine, society should be prepared for the changes that will follow the introduction of printed pharmaceuticals.

The role of cocoa as a cigarette additive: opportunities for product regulation.

PubMed

Sokol, Natasha A; Kennedy, Ryan David; Connolly, Gregory N

2014-07-01

The 2009 Family Smoking Prevention and Tobacco Control Act prohibited the use of characterizing flavors in cigarettes; however, some of these flavors are still used in cigarettes at varying levels. We reviewed tobacco industry internal documents to investigate the role of one of these flavors, cocoa, with the objective of understanding its relationship to sensory and risk perception, promotion of dependence, and enhancement of attractiveness and acceptability. We used the Legacy Tobacco Documents Library to identify documents relevant to our research questions. Initial search terms were generated following an examination of published literature on cocoa, other cigarette additives, and sensory and risk perception. Further research questions and search terms were generated based on review of documents generated from the initial search terms. Cocoa is widely applied to cigarettes and has been used by the tobacco industry as an additive since the early 20th century. Cocoa can alter the sensory properties of cigarette smoke, including by providing a more appealing taste and decreasing its harshness. The tobacco industry has experimented with manipulating cocoa levels as a means of achieving sensory properties that appeal to women and youth. Although cocoa is identified as a flavor on tobacco industry Web sites, it may serve other sensory purposes in cigarettes as well. Eliminating cocoa as an additive from tobacco products may affect tobacco product abuse liability by altering smokers' perceptions of product risk, and decreasing product appeal, especially among vulnerable populations. © The Author 2014. Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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

PubMed

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.

Prevalence, Virulence Genes, Antimicrobial Susceptibility, and Genetic Diversity of Staphylococcus aureus from Retail Aquatic Products in China

PubMed Central

Rong, Dongli; Wu, Qingping; Xu, Mingfang; Zhang, Jumei; Yu, Shubo

2017-01-01

Staphylococcus aureus is an important food-borne opportunistic pathogen that frequently causes severe blood and tissue infections or even fatal illnesses. Although S. aureus has been extensively studied in livestock and poultry foods in China, limited information has been reported in aquatic products. Accordingly, in this study, we aimed to characterize S. aureus in aquatic products purchased from retail markets in China. In total, 320 aquatic food samples were collected from 32 provincial capitals in China. The results showed that 119 samples (37.2%, 119/320) were positive for S. aureus by both qualitative and quantitative analyses. The contamination levels of 78.2% of samples ranged from 0.3 to 10 MPN/g, and six samples exceeded 110 MPN/g. A total of 119 S. aureus isolates from positive samples were selected to evaluate virulence factors, antibiotic resistance, and molecular characteristics. All S. aureus isolates were evaluated for the presence of 11 virulence genes by multiplex polymerase chain reaction, and α-hemolysin (hlα, 84.9%), fibronectin-binding protein A (fnbA, 79.0%), S. aureus enterotoxin E (see, 53.8%), and Panton-Valentine leucocidin (pvl, 50.4%) were identified as the major genes. These genes formed 56 different profiles, with the major profile identified as pvl-hlα-fnbA (28.6%). The antimicrobial susceptibility of all isolates was analyzed through the disk diffusion method, and the results showed high resistance to β-lactams, macrolides and tetracyclines, but susceptibility to linezolid and vancomycin. In addition, 26 sequence types (STs) were obtained via multilocus sequence typing, including seven novel STs, among which ST1 (20.2%), ST15 (18.5%), and ST188 (13.4%) were the most common STs. All the isolates were mecC negative, but nine isolates carrying mecA were evaluated by staphylococcal cassette chromosome mec (SCCmec) typing, all of which were SCCmecIII or SCCmecIV types. Isolates of SCCmecIII showed a high prevalence and were multidrug

21 CFR 1304.26 - Additional recordkeeping requirements applicable to drug products containing gamma-hydroxybutyric...

Code of Federal Regulations, 2010 CFR

2010-04-01

... to drug products containing gamma-hydroxybutyric acid. 1304.26 Section 1304.26 Food and Drugs DRUG....26 Additional recordkeeping requirements applicable to drug products containing gamma-hydroxybutyric....22, practitioners dispensing gamma-hydroxybutyric acid that is manufactured or distributed in...

MRG1, the product of a melanocyte-specific gene related gene, is a cytokine-inducible transcription factor with transformation activity

PubMed Central

Sun, Hui Bin; Zhu, Yuan Xiao; Yin, Tinggui; Sledge, George; Yang, Yu-Chung

1998-01-01

Identification of cytokine-inducible genes is imperative for determining the mechanisms of cytokine action. A cytokine-inducible gene, mrg1 [melanocyte-specific gene (msg1) related gene], was identified through mRNA differential display of interleukin (IL) 9-stimulated and unstimulated mouse helper T cells. In addition to IL-9, mrg1 can be induced by other cytokines and biological stimuli, including IL-1α, -2, -4, -6, and -11, granulocyte/macrophage colony-stimulating factor, interferon γ, platelet-derived growth factor, insulin, serum, and lipopolysaccharide in diverse cell types. The induction of mrg1 by these stimuli appears to be transient, with induction kinetics similar to other primary response genes, implicating its role in diverse biological processes. Deletion or point mutations of either the Box1 motif (binds Janus kinase 1) or the signal transducer and activator of transcription 3 binding site-containing region within the intracellular domain of the IL-9 receptor ligand binding subunit abolished or greatly reduced mrg1 induction by IL-9, suggesting that the Janus kinase/signal transducer and activator of transcription signaling pathway is required for mrg1 induction, at least in response to IL-9. Transfection of mrg1 cDNA into TS1, an IL-9-dependent mouse T cell line, converted these cells to IL-9-independent growth through a nonautocrine mechanism. Overexpression of mrg1 in Rat1 cells resulted in loss of cell contact inhibition, anchorage-independent growth in soft agar, and tumor formation in nude mice, demonstrating that mrg1 is a transforming gene. MRG1 is a transcriptional activator and may represent a founding member of an additional family of transcription factors. PMID:9811838

Production of Phloroglucinol, a Platform Chemical, in Arabidopsis using a Bacterial Gene.

PubMed

Abdel-Ghany, Salah E; Day, Irene; Heuberger, Adam L; Broeckling, Corey D; Reddy, Anireddy S N

2016-12-07

Phloroglucinol (1,3,5-trihydroxybenzene; PG) and its derivatives are phenolic compounds that are used for various industrial applications. Current methods to synthesize PG are not sustainable due to the requirement for carbon-based precursors and co-production of toxic byproducts. Here, we describe a more sustainable production of PG using plants expressing a native bacterial or a codon-optimized synthetic PhlD targeted to either the cytosol or chloroplasts. Transgenic lines were analyzed for the production of PG using gas and liquid chromatography coupled to mass spectroscopy. Phloroglucinol was produced in all transgenic lines and the line with the highest PhlD transcript level showed the most accumulation of PG. Over 80% of the produced PG was glycosylated to phlorin. Arabidopsis leaves have the machinery to glycosylate PG to form phlorin, which can be hydrolyzed enzymatically to produce PG. Furthermore, the metabolic profile of plants with PhlD in either the cytosol or chloroplasts was altered. Our results provide evidence that plants can be engineered to produce PG using a bacterial gene. Phytoproduction of PG using a bacterial gene paves the way for further genetic manipulations to enhance the level of PG with implications for the commercial production of this important platform chemical in plants.

Heliothis zea nudivirus 1 gene hhi1 induces apoptosis which is blocked by the Hz-iap2 gene and a noncoding gene, pag1.

PubMed

Wu, Yueh-Lung; Wu, Carol P; Liu, Catherine Y Y; Lee, Song-Tay; Lee, Hsiao-Ping; Chao, Yu-Chan

2011-07-01

Heliothis zea nudivirus 1 (HzNV-1 or Hz-1 virus), previously regarded as a nonoccluded baculovirus, recently has been placed in the Nudivirus genus. This virus generates HzNV-1 HindIII-I 1 (hhi1) and many other transcripts during productive viral infection; during latent viral infection, however, persistency-associated gene 1 (pag1) is the only gene expressed. In this report, we used transient expression assays to show that hhi1 can trigger strong apoptosis in transfected cells, which can be blocked, at least partially, by the inhibitor of apoptosis genes Autographa californica iap2 (Ac-iap2) and H. zea iap2 (Hz-iap2). In addition to these two genes, unexpectedly, pag1, which encodes a noncoding RNA with no detectable protein product, was found to efficiently suppress hhi1-induced apoptosis. The assay of pro-Sf-caspase-1 processing by hhi1 transfection did not detect the small P12 subunit at any of the time intervals tested, suggesting that hhi1 of HzNV-1 induces apoptosis through alternative caspase pathways.

A systems approach to model the relationship between aflatoxin gene cluster expression, environmental factors, growth and toxin production by Aspergillus flavus

PubMed Central

Abdel-Hadi, Ahmed; Schmidt-Heydt, Markus; Parra, Roberto; Geisen, Rolf; Magan, Naresh

2012-01-01

A microarray analysis was used to examine the effect of combinations of water activity (aw, 0.995–0.90) and temperature (20–42°C) on the activation of aflatoxin biosynthetic genes (30 genes) in Aspergillus flavus grown on a conducive YES (20 g yeast extract, 150 g sucrose, 1 g MgSO4·7H2O) medium. The relative expression of 10 key genes (aflF, aflD, aflE, aflM, aflO, aflP, aflQ, aflX, aflR and aflS) in the biosynthetic pathway was examined in relation to different environmental factors and phenotypic aflatoxin B1 (AFB1) production. These data, plus data on relative growth rates and AFB1 production under different aw × temperature conditions were used to develop a mixed-growth-associated product formation model. The gene expression data were normalized and then used as a linear combination of the data for all 10 genes and combined with the physical model. This was used to relate gene expression to aw and temperature conditions to predict AFB1 production. The relationship between the observed AFB1 production provided a good linear regression fit to the predicted production based in the model. The model was then validated by examining datasets outside the model fitting conditions used (37°C, 40°C and different aw levels). The relationship between structural genes (aflD, aflM) in the biosynthetic pathway and the regulatory genes (aflS, aflJ) was examined in relation to aw and temperature by developing ternary diagrams of relative expression. These findings are important in developing a more integrated systems approach by combining gene expression, ecophysiological influences and growth data to predict mycotoxin production. This could help in developing a more targeted approach to develop prevention strategies to control such carcinogenic natural metabolites that are prevalent in many staple food products. The model could also be used to predict the impact of climate change on toxin production. PMID:21880616

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

PubMed

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.

Pro-inflammatory NF-κB and early growth response gene 1 regulate epithelial barrier disruption by food additive carrageenan in human intestinal epithelial cells.

PubMed

Choi, Hye Jin; Kim, Juil; Park, Seong-Hwan; Do, Kee Hun; Yang, Hyun; Moon, Yuseok

2012-06-20

The widely used food additive carrageenan (CGN) has been shown to induce intestinal inflammation, ulcerative colitis-like symptoms, or neoplasm in the gut epithelia in animal models, which are also clinical features of human inflammatory bowel disease. In this study, the effects of CGN on pro-inflammatory transcription factors NF-κB and early growth response gene 1 product (EGR-1) were evaluated in terms of human intestinal epithelial barrier integrity. Both pro-inflammatory transcription factors were elevated by CGN and only NF-κB activation was shown to be involved in the induction of pro-inflammatory cytokine interleukin-8. Moreover, the integrity of the in vitro epithelial monolayer under the CGN insult was maintained by both activated pro-inflammatory transcription factors NF-κB and EGR-1. Suppression of NF-κB or EGR-1 aggravated barrier disruption by CGN, which was associated with the reduced gene expression of tight junction component zonula occludens 1 and its irregular localization in the epithelial monolayer. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Discovering gene annotations in biomedical text databases.

PubMed

Cakmak, Ali; Ozsoyoglu, Gultekin

2008-03-06

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. 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. 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 pattern occurrences with similar semantics. Relatively

75 FR 33824 - Pharmaceutical Products and Chemical Intermediates, Fourth Review: Advice Concerning the Addition...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-06-15

... production of pharmaceuticals (pharmaceuticals zero-for-zero initiative) and to conduct periodic reviews to... eliminated duties on additional pharmaceutical items. The USTR indicated that participants in the zero- for-zero initiative are conducting a fourth review to determine if products can be added to the initiative...

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

PubMed

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.

The Crc protein participates in down-regulation of the Lon gene to promote rhamnolipid production and rhl quorum sensing in Pseudomonas aeruginosa.

PubMed

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.

Biofilm formation, phenotypic production of cellulose and gene expression in Salmonella enterica decrease under anaerobic conditions.

PubMed

Lamas, A; Miranda, J M; Vázquez, B; Cepeda, A; Franco, C M

2016-12-05

Salmonella enterica subsp. enterica is one of the main food-borne pathogens. This microorganism combines an aerobic life outside the host with an anaerobic life within the host. One of the main concerns related to S. enterica is biofilm formation and cellulose production. In this study, biofilm formation, morphotype, cellulose production and transcription of biofilm and quorum sensing-related genes of 11 S. enterica strains were tested under three different conditions: aerobiosis, microaerobiosis, and anaerobiosis. The results showed an influence of oxygen levels on biofilm production. Biofilm formation was significantly higher (P<0.05) in aerobiosis than in microaerobiosis and anaerobiosis. Cellulose production and RDAR (red, dry, and rough) were expressed only in aerobiosis. In microaerobiosis, the strains expressed the SAW (smooth and white) morphotype, while in anaerobiosis the colonies appeared small and red. The expression of genes involved in cellulose synthesis (csgD and adrA) and quorum sensing (sdiA and luxS) was reduced in microaerobiosis and anaerobiosis in all S. enterica strains tested. This gene expression levels were less reduced in S. Typhimurium and S. Enteritidis compared to the tested serotypes. There was a relationship between the expression of biofilm and quorum sensing-related genes. Thus, the results from this study indicate that biofilm formation and cellulose production are highly influenced by atmospheric conditions. This must be taken into account as contamination with these bacteria can occur during food processing under vacuum or modified atmospheres. Copyright © 2016 Elsevier B.V. All rights reserved.

Primary hyperoxaluria type 1: update and additional mutation analysis of the AGXT gene.

PubMed

Williams, Emma L; Acquaviva, Cecile; Amoroso, Antonio; Chevalier, Francoise; Coulter-Mackie, Marion; Monico, Carla G; Giachino, Daniela; Owen, Tricia; Robbiano, Angela; Salido, Eduardo; Waterham, Hans; Rumsby, Gill

2009-06-01

Primary hyperoxaluria type 1 (PH1) is an autosomal recessive, inherited disorder of glyoxylate metabolism arising from a deficiency of the alanine:glyoxylate aminotransferase (AGT) enzyme, encoded by the AGXT gene. The disease is manifested by excessive endogenous oxalate production, which leads to impaired renal function and associated morbidity. At least 146 mutations have now been described, 50 of which are newly reported here. The mutations, which occur along the length of the AGXT gene, are predominantly single-nucleotide substitutions (75%), 73 are missense, 19 nonsense, and 18 splice mutations; but 36 major and minor deletions and insertions are also included. There is little association of mutation with ethnicity, the most obvious exception being the p.Ile244Thr mutation, which appears to have North African/Spanish origins. A common, polymorphic variant encoding leucine at codon 11, the so-called minor allele, has significantly lower catalytic activity in vitro, and has a higher frequency in PH1 compared to the rest of the population. This polymorphism influences enzyme targeting in the presence of the most common Gly170Arg mutation and potentiates the effect of several other pathological sequence variants. This review discusses the spectrum of AGXT mutations and polymorphisms, their clinical significance, and their diagnostic relevance.

A novel riboregulator switch system of gene expression for enhanced microbial production of succinic acid.

PubMed

Wang, Jing; Wang, Haoyuan; Yang, Le; Lv, Liping; Zhang, Zhe; Ren, Bin; Dong, Lichun; Li, Ning

2018-04-01

In this paper, a novel riboregulator Switch System of Gene Expression including an OFF-TO-ON switch and an ON-TO-OFF switch was designed to regulate the expression state of target genes between "ON" and "OFF" by switching the identifiability of ribosome recognition site (RBS) based on the thermodynamic stability of different RNA-RNA hybridizations between RBS and small noncoding RNAs. The proposed riboregulator switch system was employed for the fermentative production of succinic acid using an engineered strain of E. coli JW1021, during which the expression of mgtC gene was controlled at "ON" state and that of pepc and ecaA genes were controlled at the "OFF" state in the lag phase and switched to the "OFF" and "ON" state once the strain enters the logarithmic phase. The results showed that using the strain of JW1021, the yield and productivity of succinic acid can reach 0.91 g g -1 and 3.25 g L -1  h -1 , respectively, much higher than those using the strains without harboring the riboregulator switch system.

Association of methionine synthase gene polymorphisms with wool production and quality traits in Chinese Merino population.

PubMed

Rong, E G; Yang, H; Zhang, Z W; Wang, Z P; Yan, X H; Li, H; Wang, N

2015-10-01

Methionine synthase (MTR) plays a crucial role in maintaining homeostasis of intracellular methionine, folate, and homocysteine, and its activity correlates with DNA methylation in many mammalian tissues. Our previous genomewide association study identified that 1 SNP located in the gene was associated with several wool production and quality traits in Chinese Merino. To confirm the potential involvement of the gene in sheep wool production and quality traits, we performed sheep tissue expression profiling, SNP detection, and association analysis with sheep wool production and quality traits. The semiquantitative reverse transcription PCR analysis showed that the gene was differentially expressed in skin from Merino and Kazak sheep. The sequencing analysis identified a total of 13 SNP in the gene from Chinese Merino sheep. Comparison of the allele frequencies revealed that these 13 identified SNP were significantly different among the 6 tested Chinese Merino strains ( < 0.001). Linkage disequilibrium analysis showed that SNP 3 to 11 were strongly linked in a single haplotype block in the tested population. Association analysis showed that SNP 2 to 11 were significantly associated with the average wool fiber diameter and the fineness SD and that SNP 4 to 11 were significantly associated with the CV of fiber diameter trait ( < 0.05). Single nucleotide polymorphism 2 and SNP 5 to 12 were weakly associated with wool crimp. Similarly, the haplotypes derived from these 13 identified SNP were also significantly associated with the average wool fiber diameter, fineness SD, and the CV of fiber diameter ( < 0.05). Our results suggest that is a candidate gene for sheep wool production and quality traits, and the identified SNP might be used in sheep breeding.

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

PubMed

El Khoury, Rachelle; Atoui, Ali; Verheecke, Carol; Maroun, Richard; El Khoury, Andre; Mathieu, Florence

2016-08-19

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 1 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 µL/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.

Efficient and rapid Agrobacterium-mediated genetic transformation of durum wheat (Triticum turgidum L. var. durum) using additional virulence genes.

PubMed

Wu, Huixia; Doherty, Angela; Jones, Huw D

2008-06-01

Genetic transformation of wheat, using biolistics or Agrobacterium, underpins a range of specific research methods for identifying genes and studying their function in planta. Transgenic approaches to study and modify traits in durum wheat have lagged behind those for bread wheat. Here we report the use of Agrobacterium strain AGL1, with additional vir genes housed in a helper plasmid, to transform and regenerate the durum wheat variety Ofanto. The use of the basic pSoup helper plasmid with no additional vir genes failed to generate transformants, whereas the presence of either virG542 or the 15 kb Komari fragment containing virB, virC and virG542 produced transformation efficiencies of between 0.6 and 9.7%. Of the 42 transgenic plants made, all but one (which set very few seeds) appeared morphologically normal and produced between 100 and 300 viable seeds. The transgene copy number and the segregation ratios were found to be very similar to those previously reported for bread wheat. We believe that this is the first report describing successful genetic transformation of tetraploid durum wheat (Triticum turgidum L. var. durum) mediated by Agrobacterium tumefaciens using immature embryos as the explant.

The Rice Tungro Bacilliform Virus Gene II Product Interacts with the Coat Protein Domain of the Viral Gene III Polyprotein

PubMed Central

Herzog, Etienne; Guerra-Peraza, Orlene; Hohn, Thomas

2000-01-01

Rice tungro bacilliform virus (RTBV) is a plant pararetrovirus whose DNA genome contains four genes encoding three proteins and a large polyprotein. The function of most of the viral proteins is still unknown. To investigate the role of the gene II product (P2), we searched for interactions between this protein and other RTBV proteins. P2 was shown to interact with the coat protein (CP) domain of the viral gene III polyprotein (P3) both in the yeast two-hybrid system and in vitro. Domains involved in the P2-CP association have been identified and mapped on both proteins. To determine the importance of this interaction for viral multiplication, the infectivity of RTBV gene II mutants was investigated by agroinoculation of rice plants. The results showed that virus viability correlates with the ability of P2 to interact with the CP domain of P3. This study suggests that P2 could participate in RTBV capsid assembly. PMID:10666237

When monoclonal antibodies are not monospecific: Hybridomas frequently express additional functional variable regions

PubMed Central

Bradbury, Andrew R. M.; Trinklein, Nathan D.; Wilkinson, Ian C.; Tandon, Atul K.; Anderson, Stephen; Bladen, Catherine L.; Jones, Brittany; Aldred, Shelley Force; Bestagno, Marco; Burrone, Oscar; Maynard, Jennifer; Ferrara, Fortunato; Görnemann, Janina; Glanville, Jacob; Wolf, Philipp; Frenzel, Andre; Wong, Julin; Koh, Xin Yu; Eng, Hui-Yan; Lane, David; Lefranc, Marie-Paule; Clark, Mike

2018-01-01

ABSTRACT Monoclonal antibodies are commonly assumed to be monospecific, but anecdotal studies have reported genetic diversity in antibody heavy chain and light chain genes found within individual hybridomas. As the prevalence of such diversity has never been explored, we analyzed 185 random hybridomas, in a large multicenter dataset. The hybridomas analyzed were not biased towards those with cloning difficulties or known to have additional chains. Of the hybridomas we evaluated, 126 (68.1%) contained no additional productive chains, while the remaining 59 (31.9%) contained one or more additional productive heavy or light chains. The expression of additional chains degraded properties of the antibodies, including specificity, binding signal and/or signal-to-noise ratio, as determined by enzyme-linked immunosorbent assay and immunohistochemistry. The most abundant mRNA transcripts found in a hybridoma cell line did not necessarily encode the antibody chains providing the correct specificity. Consequently, when cloning antibody genes, functional validation of all possible VH and VL combinations is required to identify those with the highest affinity and lowest cross-reactivity. These findings, reflecting the current state of hybridomas used in research, reiterate the importance of using sequence-defined recombinant antibodies for research or diagnostic use. PMID:29485921

Streptomyces artemisiae MCCB 248 isolated from Arctic fjord sediments has unique PKS and NRPS biosynthetic genes and produces potential new anticancer natural products.

PubMed

Dhaneesha, M; Benjamin Naman, C; Krishnan, K P; Sinha, Rupesh Kumar; Jayesh, P; Joseph, Valsamma; Bright Singh, I S; Gerwick, William H; Sajeevan, T P

2017-05-01

After screening marine actinomycetes isolated from sediment samples collected from the Arctic fjord Kongsfjorden for potential anticancer activity, an isolate identified as Streptomyces artemisiae MCCB 248 exhibited promising results against the NCI-H460 human lung cancer cell line. H460 cells treated with the ethyl acetate extract of strain MCCB 248 and stained with Hoechst 33342 showed clear signs of apoptosis, including shrinkage of the cell nucleus, DNA fragmentation and chromatin condensation. Further to this treated cells showed indications of early apoptotic cell death, including a significant proportion of Annexin V positive staining and evidence of DNA damage as observed in the TUNEL assay. Amplified PKS 1 and NRPS genes involved in secondary metabolite production showed only 82% similarity to known biosynthetic genes of Streptomyces, indicating the likely production of a novel secondary metabolite in this extract. Additionally, chemical dereplication efforts using LC-MS/MS molecular networking suggested the presence of a series of undescribed tetraene polyols. Taken together, these results revealed that this Arctic S. artemisiae strain MCCB 248 is a promising candidate for natural products drug discovery and genome mining for potential anticancer agents.

The light gene of Drosophila melanogaster encodes a homologue of VPS41, a yeast gene involved in cellular-protein trafficking.

PubMed

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.

Gene therapy for immune disorders: good news tempered by bad news.

PubMed

Puck, Jennifer M; Malech, Harry L

2006-04-01

After a dozen years of human gene therapy trials characterized by minimal gene correction and disappointing clinical impact, the field of gene therapy received some good news in 2000. Infants with X-linked severe combined immunodeficiency who received retroviral gene addition to cells from their bone marrow developed impressive immune reconstitution. During the following 2 years, additional patients were treated and the news was even better-babies receiving gene therapy had sustained T-cell production and in several cases developed better cell function than most patients treated with standard bone marrow transplants. Unfortunately, bad news followed. Three of the patients experienced leukemic T-cell expansions, found to be associated with retroviral insertions into genomic DNA. Where does the field stand today?

Analysis of the neuronal marker protein gene product 9.5 in internal limiting membranes after indocyanine-green assisted peeling.

PubMed

Peters, Swaantje; Tatar, Olcay; Spitzer, Martin S; Szurman, Peter; Aisenbrey, Sabine; Lüke, Matthias; Adam, Annemarie; Yoeruek, Efdal; Grisanti, Salvatore

2009-02-01

Indocyanine green-assisted internal limiting membrane (ILM) peeling was suspected to disrupt the innermost layer of the neural retina. We examined whether surgically excised specimens contain remnants of neuronal tissue. Ten patients with macular hole underwent pars plana vitrectomy and indocyanine green-assisted ILM peeling. A total of 0.1 mL of a 0.5% indocyanine green solution was applied for 15 seconds. The ILM specimens were prepared for immunohistochemistry, using a polyclonal antibody against protein gene product 9.5. Protein gene product 9.5 is a pan-neuronal marker labeling human neuronal cells. Appropriate controls to show selectivity of the antibody were performed on neuronal tissue of donor eyes. One ILM was prepared for electron microscopy. A selective expression of protein gene product 9.5 was found in neuronal fibers of the retina and optic nerve of donor eyes. Only 1 of the 10 surgical ILM specimens showed a minimal focal positivity for protein gene product 9.5. No neuronal tissue was detected on the ILM by electron microscopy. Focal expression of protein gene product 9.5 in only 1 of 10 surgical ILM specimens argues against a general indocyanine green-related disruption of the innermost retinal layers. However, higher concentrations of the dye, longer incubation times or different solvents than used in this study may lead to different results.

CO 2 Reduction Selective for C ≥2 Products on Polycrystalline Copper with N-Substituted Pyridinium Additives

DOE PAGES

Han, Zhiji; Kortlever, Ruud; Chen, Hsiang -Yun; ...

2017-07-21

Electrocatalytic CO 2 reduction to generate multicarbon products is of interest for applications in artificial photosynthetic schemes. This is a particularly attractive goal for CO 2 reduction by copper electrodes, where a broad range of hydrocarbon products can be generated but where selectivity for C–C coupled products relative to CH 4 and H 2 remains an impediment. Herein we report a simple yet highly selective catalytic system for CO 2 reduction to C ≥2 hydrocarbons on a polycrystalline Cu electrode in bicarbonate aqueous solution that uses N-substituted pyridinium additives. Selectivities of 70–80% for C 2 and C 3 products withmore » a hydrocarbon ratio of C ≥2/CH 4 significantly greater than 100 have been observed with several additives. 13C-labeling studies verify CO 2 to be the sole carbon source in the C ≥2 hydrocarbons produced. Upon electroreduction, the N-substituted pyridinium additives lead to film deposition on the Cu electrode, identified in one case as the reductive coupling product of N-arylpyridinium. As a result, product selectivity can also be tuned from C ≥2 species to H 2 (~90%) while suppressing methane with certain N-heterocyclic additives.« less

CO 2 Reduction Selective for C ≥2 Products on Polycrystalline Copper with N-Substituted Pyridinium Additives

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Zhiji; Kortlever, Ruud; Chen, Hsiang -Yun

Electrocatalytic CO 2 reduction to generate multicarbon products is of interest for applications in artificial photosynthetic schemes. This is a particularly attractive goal for CO 2 reduction by copper electrodes, where a broad range of hydrocarbon products can be generated but where selectivity for C–C coupled products relative to CH 4 and H 2 remains an impediment. Herein we report a simple yet highly selective catalytic system for CO 2 reduction to C ≥2 hydrocarbons on a polycrystalline Cu electrode in bicarbonate aqueous solution that uses N-substituted pyridinium additives. Selectivities of 70–80% for C 2 and C 3 products withmore » a hydrocarbon ratio of C ≥2/CH 4 significantly greater than 100 have been observed with several additives. 13C-labeling studies verify CO 2 to be the sole carbon source in the C ≥2 hydrocarbons produced. Upon electroreduction, the N-substituted pyridinium additives lead to film deposition on the Cu electrode, identified in one case as the reductive coupling product of N-arylpyridinium. As a result, product selectivity can also be tuned from C ≥2 species to H 2 (~90%) while suppressing methane with certain N-heterocyclic additives.« less

Dropwise additive manufacturing of pharmaceutical products for melt-based dosage forms.

PubMed

Içten, Elçin; Giridhar, Arun; Taylor, Lynne S; Nagy, Zoltan K; Reklaitis, Gintaras V

2015-05-01

The US Food and Drug Administration introduced the quality by design approach and process analytical technology guidance to encourage innovation and efficiency in pharmaceutical development, manufacturing, and quality assurance. As part of this renewed emphasis on the improvement of manufacturing, the pharmaceutical industry has begun to develop more efficient production processes with more intensive use of online measurement and sensing, real-time quality control, and process control tools. Here, we present dropwise additive manufacturing of pharmaceutical products (DAMPP) as an alternative to conventional pharmaceutical manufacturing methods. This mini-manufacturing process for the production of pharmaceuticals utilizes drop on demand printing technology for automated and controlled deposition of melt-based formulations onto edible substrates. The advantages of drop-on-demand technology, including reproducible production of small droplets, adjustable drop sizing, high placement accuracy, and flexible use of different formulations, enable production of individualized dosing even for low-dose and high-potency drugs. In this work, DAMPP is used to produce solid oral dosage forms from hot melts of an active pharmaceutical ingredient and a polymer. The dosage forms are analyzed to show the reproducibility of dosing and the dissolution behavior of different formulations. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

Indications of Transformation Products from Hydraulic Fracturing Additives in Shale-Gas Wastewater.

PubMed

Hoelzer, Kathrin; Sumner, Andrew J; Karatum, Osman; Nelson, Robert K; Drollette, Brian D; O'Connor, Megan P; D'Ambro, Emma L; Getzinger, Gordon J; Ferguson, P Lee; Reddy, Christopher M; Elsner, Martin; Plata, Desiree L

2016-08-02

Unconventional natural gas development (UNGD) generates large volumes of wastewater, the detailed composition of which must be known for adequate risk assessment and treatment. In particular, transformation products of geogenic compounds and disclosed additives have not been described. This study investigated six Fayetteville Shale wastewater samples for organic composition using a suite of one- and two-dimensional gas chromatographic techniques to capture a broad distribution of chemical structures. Following the application of strict compound-identification-confidence criteria, we classified compounds according to their putative origin. Samples displayed distinct chemical distributions composed of typical geogenic substances (hydrocarbons and hopane biomarkers), disclosed UNGD additives (e.g., hydrocarbons, phthalates such as diisobutyl phthalate, and radical initiators such as azobis(isobutyronitrile)), and undisclosed compounds (e.g., halogenated hydrocarbons, such as 2-bromohexane or 4-bromoheptane). Undisclosed chloromethyl alkanoates (chloromethyl propanoate, pentanoate, and octanoate) were identified as potential delayed acids (i.e., those that release acidic moieties only after hydrolytic cleavage, the rate of which could be potentially controlled), suggesting they were deliberately introduced to react in the subsurface. In contrast, the identification of halogenated methanes and acetones suggested that those compounds were formed as unintended byproducts. Our study highlights the possibility that UNGD operations generate transformation products and underscores the value of disclosing additives injected into the subsurface.

Improvement of bioinsecticides production through adaptation of Bacillus thuringiensis cells to heat treatment and NaCl addition.

PubMed

Ghribi, D; Zouari, N; Jaoua, S

2005-01-01

The present work aimed to increase yields of delta-endotoxin production through adaptation of Bacillus thuringiensis cells to heat shock and sodium chloride and to investigate their involvements in bioinsecticides production improvement. Growing B. thuringiensis cells were heat treated after different incubation times to study the response of the adaptative surviving cells in terms of delta-endotoxin synthesis. Similarly, adaptation of B. thuringiensis cells to sodium chloride was investigated. Adaptation to combined stressors was also evaluated. When applied separately in the glucose-based medium, 20-min heat treatment of 6-h-old cultures and addition of 7 g l(-1) NaCl at the beginning of the incubation gave respectively 38 and 27% delta-endotoxin production improvements. Heat shock improved toxin synthesis yields, while NaCl addition improved delta-endotoxin production by increasing the spore titres without significant effect on toxin synthesis yields. Cumulative improvements (66%) were obtained by combination of the two stressors at the conditions previously established for each one. Interestingly, when the similar approach was conducted by using the large scale production medium based on gruel and fish meal, 17, 8 and 29% delta-endotoxin production improvements were respectively, obtained with heat shock, NaCl and combined stressors. Heat treatment of vegetative B. thuringiensis cells and NaCl addition to the culture media improved bioinsecticides production. Heat treatment increased toxin synthesis yields, while addition of NaCl increased biomass production yields. Cumulative improvements of 66 and 29% were obtained in glucose and economic production media, respectively. Overproduction of bioinsecticides by B. thuringiensis could be obtained by the combination of heat treatment of vegetative cells and addition of NaCl to the culture medium. This should contribute to a significant reduction of the cost of B. thuringiensis bioinsecticides production and

Lethality of mice bearing a knockout of the Ngly1-gene is partially rescued by the additional deletion of the Engase gene

PubMed Central

Fujihira, Haruhiko; Masahara-Negishi, Yuki; Tamura, Masaru; Huang, Chengcheng; Harada, Yoichiro; Wakana, Shigeharu; Takakura, Daisuke; Kawasaki, Nana; Taniguchi, Naoyuki; Kondoh, Gen; Yamashita, Tadashi; Funakoshi, Yoko; Suzuki, Tadashi

2017-01-01

The cytoplasmic peptide:N-glycanase (Ngly1 in mammals) is a de-N-glycosylating enzyme that is highly conserved among eukaryotes. It was recently reported that subjects harboring mutations in the NGLY1 gene exhibited severe systemic symptoms (NGLY1-deficiency). While the enzyme obviously has a critical role in mammals, its precise function remains unclear. In this study, we analyzed Ngly1-deficient mice and found that they are embryonic lethal in C57BL/6 background. Surprisingly, the additional deletion of the gene encoding endo-β-N-acetylglucosaminidase (Engase), which is another de-N-glycosylating enzyme but leaves a single GlcNAc at glycosylated Asn residues, resulted in the partial rescue of the lethality of the Ngly1-deficient mice. Additionally, we also found that a change in the genetic background of C57BL/6 mice, produced by crossing the mice with an outbred mouse strain (ICR) could partially rescue the embryonic lethality of Ngly1-deficient mice. Viable Ngly1-deficient mice in a C57BL/6 and ICR mixed background, however, showed a very severe phenotype reminiscent of the symptoms of NGLY1-deficiency subjects. Again, many of those defects were strongly suppressed by the additional deletion of Engase in the C57BL/6 and ICR mixed background. The defects observed in Ngly1/Engase-deficient mice (C57BL/6 background) and Ngly1-deficient mice (C57BL/6 and ICR mixed background) closely resembled some of the symptoms of patients with an NGLY1-deficiency. These observations strongly suggest that the Ngly1- or Ngly1/Engase-deficient mice could serve as a valuable animal model for studies related to the pathogenesis of the NGLY1-deficiency, and that cytoplasmic ENGase represents one of the potential therapeutic targets for this genetic disorder. PMID:28426790

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

PubMed Central

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

2014-01-01

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

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

USGS Publications Warehouse

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

Detection of classical enterotoxins and identification of enterotoxin genes in Staphylococcus aureus from milk and dairy products.

PubMed

Morandi, S; Brasca, M; Lodi, R; Cremonesi, P; Castiglioni, B

2007-09-20

Milk and dairy products are frequently contaminated with enterotoxigenic Staphylococcus aureus, which is often involved in staphylococcal food poisoning. The distribution of genes encoding staphylococcal enterotoxins (SE) in S. aureus isolated from bovine, goat, sheep and buffalo milk and dairy products was verified by the presence of the corresponding SE production. A total of 112 strains of S. aureus were tested for SE production by immuno-enzymatic (SEA-SEE) and reversed passive latex agglutination (SEA-SED) methods, while multiplex-PCR was applied for SE genes (sea, sec, sed, seg, seh, sei, sej and sel). Of the total strains studied, 67% were detected to have some SE genes (se), but only 52% produced a detectable amount of the classic antigenic SE types. The bovine isolates frequently had enterotoxin SEA, SED and sej, while SEC and sel predominated in the goat and sheep strains. The results demonstrated (i) marked enterotoxigenic S. aureus strain variations, in accordance with strain origin and (ii) the two methods resulted in different information but concurred on the risk of foodstuff infection by S. aureus.

Transcriptome analysis of genes related to resistance against powdery mildew in wheat-Thinopyrum alien addition disomic line germplasm SN6306.

PubMed

Li, Quanquan; Niu, Zubiao; Bao, Yinguang; Tian, Qiuju; Wang, Honggang; Kong, Lingrang; Feng, Deshun

2016-09-15

Wheat powdery mildew, which is mainly caused by Blumeria graminis f. sp. tritici (Bgt), seriously damages wheat production. The wheat-Thinopyrum intermedium alien addition disomic line germplasm SN6306, being one of the important sources of genes for wheat resistance, is highly resistant to Bgt E09 and to many other powdery mildew physiological races. However, knowledge on the resistance mechanism of SN6306 remains limited. Our study employed high-throughput RNA sequencing based on next-generation sequencing technology (Illumina) to obtain an overview of the transcriptome characteristics of SN6306 and its parent wheat Yannong 15 (YN15) during Bgt infection. The sequencing generated 104,773 unigenes, 9909 of which showed varied expression levels. Among the 9909 unigenes, 1678 unigenes showed 0 reads in YN15. The expression levels in Bgt-inoculated SN6306 and YN15 of exactly 39 unigenes that showed 0 or considerably low reads in YN15 were validated to identify the genes involved in Bgt resistance. Among the 39 unigenes, 12 unigenes were upregulated in SN6306 by 3-45 times. These unigenes mainly encoded kinase, synthase, proteases, and signal transduction proteins, which may play an important role in the resistance against Bgt. To confirm whether the unigenes that showed 0 reads in YN15 are really unique to SN6306, 8 unigenes were cloned and sequenced. Results showed that the selected unigenes are more similar to SN6306 and Th. intermedium than to the wheat cultivar YN15. The sequencing results further confirmed that the unigenes showing 0 reads in YN15 are unique to SN6306 and are most likely derived from Th. intermedium (Host) Nevski. Thus, the genes from Th. intermedium most probably conferred the resistance of SN6306 to Bgt. Copyright © 2016 Elsevier B.V. All rights reserved.

Co-expression of Exo-inulinase and Endo-inulinase Genes in the Oleaginous Yeast Yarrowia lipolytica for Efficient Single Cell Oil Production from Inulin.

PubMed

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.

Deletion of the hfsB gene increases ethanol production in Thermoanaerobacterium saccharolyticum and several other thermophilic anaerobic bacteria.

PubMed

Eminoğlu, Ayşenur; Murphy, Sean Jean-Loup; Maloney, Marybeth; Lanahan, Anthony; Giannone, Richard J; Hettich, Robert L; Tripathi, Shital A; Beldüz, Ali Osman; Lynd, Lee R; Olson, Daniel G

2017-01-01

With the discovery of interspecies hydrogen transfer in the late 1960s (Bryant et al. in Arch Microbiol 59:20-31, 1967), it was shown that reducing the partial pressure of hydrogen could cause mixed acid fermenting organisms to produce acetate at the expense of ethanol. Hydrogen and ethanol are both more reduced than glucose. Thus there is a tradeoff between production of these compounds imposed by electron balancing requirements; however, the mechanism is not fully known. Deletion of the hfsA or B subunits resulted in a roughly 1.8-fold increase in ethanol yield. The increase in ethanol production appears to be associated with an increase in alcohol dehydrogenase activity, which appears to be due, at least in part, to increased expression of the adhE gene, and may suggest a regulatory linkage between hfsB and adhE . We studied this system most intensively in the organism Thermoanaerobacterium saccharolyticum ; however, deletion of hfsB also increases ethanol production in other thermophilic bacteria suggesting that this could be used as a general technique for engineering thermophilic bacteria for improved ethanol production in organisms with hfs -type hydrogenases. Since its discovery by Shaw et al. (JAMA 191:6457-64, 2009), the hfs hydrogenase has been suspected to act as a regulator due to the presence of a PAS domain. We provide additional support for the presence of a regulatory phenomenon. In addition, we find a practical application for this scientific insight, namely increasing ethanol yield in strains that are of interest for ethanol production from cellulose or hemicellulose. In two of these organisms ( T. xylanolyticum and T. thermosaccharolyticum ), the ethanol yields are the highest reported to date.

EMEA and Gene Therapy Medicinal Products Development in the European Union

PubMed Central

2003-01-01

The evaluation of quality, safety, and efficacy of medicinal products by the European Medicines Evaluation Agency (EMEA) via the centralized procedure is the only available regulatory procedure for obtaining marketing authorization for gene therapy (GT) medicinal products in the European Union. The responsibility for the authorization of clinical trials remains with the national competent authorities (NCA) acting in a harmonized framework from the scientific viewpoint. With the entry into force of a new directive on good clinical practice implementation in clinical trials as of 1 May 2004, procedural aspects will also be harmonized at EU level. Scientifically sound development of medicinal products is the key for the successful registration of dossiers and for contributing to the promotion and protection of public health. The objective of this paper is to introduce the EMEA regulatory processes and scientific activities relevant to GT medicinal products. PMID:12686717

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

PubMed

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.

The Treacher Collins syndrome (TCOF1) gene product is involved in pre-rRNA methylation.

PubMed

Gonzales, Bianca; Henning, Dale; So, Rolando B; Dixon, Jill; Dixon, Michael J; Valdez, Benigno C

2005-07-15

Treacher Collins syndrome (TCS) is characterized by defects in craniofacial development, which results from mutations in the TCOF1 gene. TCOF1 encodes the nucleolar phosphoprotein treacle, which interacts with upstream binding factor (UBF) and affects transcription of the ribosomal DNA gene. The present study shows participation of treacle in the 2'-O-methylation of pre-rRNA. Antisense-mediated down-regulation of treacle expression in Xenopus laevis oocytes reduced 2'-O-methylation of pre-rRNA. Analysis of RNA isolated from wild-type and Tcof1+/- heterozygous mice embryos from strains that exhibit a lethal phenotype showed significant reduction in 2'-O-methylation at nucleotide C463 of 18S rRNA. The level of pseudouridylation of U1642 of 18S rRNA from the same RNA samples was not affected suggesting specificity. There is no significant difference in rRNA methylation between wild-type and heterozygous embryos of DBA x BALB/c mice, which have no obvious craniofacial phenotype. The function of treacle in pre-rRNA methylation is most likely mediated by its direct physical interaction with NOP56, a component of the ribonucleoprotein methylation complex. Although treacle co-localizes with UBF throughout mitosis, it co-localizes with NOP56 and fibrillarin, a putative methyl transferase, only during telophase when rDNA gene transcription and pre-rRNA methylation are known to commence. These observations suggest that treacle might link RNA polymerase I-catalyzed transcription and post-transcriptional modification of pre-rRNA. We hypothesize that haploinsufficiency of treacle in TCS patients results in inhibition of production of properly modified mature rRNA in addition to inhibition of rDNA gene transcription, which consequently affects proliferation and proper differentiation of specific embryonic cells during development.

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

PubMed

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

The sps Gene Products Affect the Germination, Hydrophobicity, and Protein Adsorption of Bacillus subtilis Spores

PubMed Central

Cangiano, Giuseppina; Sirec, Teja; Panarella, Cristina; Isticato, Rachele; Baccigalupi, Loredana; De Felice, Maurilio

2014-01-01

The multilayered surface of the Bacillus subtilis spore is composed of proteins and glycans. While over 70 different proteins have been identified as surface components, carbohydrates associated with the spore surface have not been characterized in detail yet. Bioinformatic data suggest that the 11 products of the sps operon are involved in the synthesis of polysaccharides present on the spore surface, but an experimental validation is available only for the four distal genes of the operon. Here, we report a transcriptional analysis of the sps operon and a functional study performed by constructing and analyzing two null mutants lacking either all or only the promoter-proximal gene of the operon. Our results show that both sps mutant spores apparently have normal coat and crust but have a small germination defect and are more hydrophobic than wild-type spores. We also show that spores lacking all Sps proteins are highly adhesive and form extensive clumps. In addition, sps mutant spores have an increased efficiency in adsorbing a heterologous enzyme, suggesting that hydrophobic force is a major determinant of spore adsorption and indicating that a deep understanding of the surface properties of the spore is essential for its full development as a surface display platform. PMID:25239894

A Brain Membrane Protein Similar to the Rat src Gene Product

NASA Astrophysics Data System (ADS)

Scheinberg, David A.; Strand, Mette

1981-01-01

We report the purification to homogeneity of a 20,000-dalton, transformation-related, rat cell membrane protein. This protein, p20, was originally identified in preparations of a defective woolly monkey leukemia virus pseudotype of Kirsten sarcoma virus. The chromatographically purified p20 was an acidic hydrophobic protein, capable of specifically binding GTP (dissociation constant = 15 μ M). This nucleotide binding property and other previously reported characteristics were similar to properties ascribed to the Harvey sarcoma virus src gene product. p20 also appeared similar to this src gene product when immunoprecipitates of both proteins were directly compared by one- and two-dimensional NaDodSO4 gel electrophoreses. However, the proteins were not identical, because their tryptic maps differed. Using a competition radioimmunoassay, we have measured the concentration of p20 in cells, viruses, and rat tissues: p20 was not encoded by rat sarcoma viruses because it was increased only slightly after Kirsten sarcoma virus transformation of rat cells and was not increased in nonrat cells transformed by the Kirsten or Harvey sarcoma virus. Remarkably, of 10 rat tissues examined, p20 was found predominantly in brain, specifically in the membranes.

The Symbiotic Performance of Chickpea Rhizobia Can Be Improved by Additional Copies of the clpB Chaperone Gene.

PubMed

Paço, Ana; Brígido, Clarisse; Alexandre, Ana; Mateos, Pedro F; Oliveira, Solange

2016-01-01

The ClpB chaperone is known to be involved in bacterial stress response. Moreover, recent studies suggest that this protein has also a role in the chickpea-rhizobia symbiosis. In order to improve both stress tolerance and symbiotic performance of a chickpea microsymbiont, the Mesorhizobium mediterraneum UPM-Ca36T strain was genetically transformed with pPHU231 containing an extra-copy of the clpB gene. To investigate if the clpB-transformed strain displays an improved stress tolerance, bacterial growth was evaluated under heat and acid stress conditions. In addition, the effect of the extra-copies of the clpB gene in the symbiotic performance was evaluated using plant growth assays (hydroponic and pot trials). The clpB-transformed strain is more tolerant to heat shock than the strain transformed with pPHU231, supporting the involvement of ClpB in rhizobia heat shock tolerance. Both plant growth assays showed that ClpB has an important role in chickpea-rhizobia symbiosis. The nodulation kinetics analysis showed a higher rate of nodule appearance with the clpB-transformed strain. This strain also induced a greater number of nodules and, more notably, its symbiotic effectiveness increased ~60% at pH5 and 83% at pH7, compared to the wild-type strain. Furthermore, a higher frequency of root hair curling was also observed in plants inoculated with the clpB-transformed strain, compared to the wild-type strain. The superior root hair curling induction, nodulation ability and symbiotic effectiveness of the clpB-transformed strain may be explained by an increased expression of symbiosis genes. Indeed, higher transcript levels of the nodulation genes nodA and nodC (~3 folds) were detected in the clpB-transformed strain. The improvement of rhizobia by addition of extra-copies of the clpB gene may be a promising strategy to obtain strains with enhanced stress tolerance and symbiotic effectiveness, thus contributing to their success as crop inoculants, particularly under

The Symbiotic Performance of Chickpea Rhizobia Can Be Improved by Additional Copies of the clpB Chaperone Gene

PubMed Central

Paço, Ana; Brígido, Clarisse; Alexandre, Ana; Mateos, Pedro F.; Oliveira, Solange

2016-01-01

The ClpB chaperone is known to be involved in bacterial stress response. Moreover, recent studies suggest that this protein has also a role in the chickpea-rhizobia symbiosis. In order to improve both stress tolerance and symbiotic performance of a chickpea microsymbiont, the Mesorhizobium mediterraneum UPM-Ca36T strain was genetically transformed with pPHU231 containing an extra-copy of the clpB gene. To investigate if the clpB-transformed strain displays an improved stress tolerance, bacterial growth was evaluated under heat and acid stress conditions. In addition, the effect of the extra-copies of the clpB gene in the symbiotic performance was evaluated using plant growth assays (hydroponic and pot trials). The clpB-transformed strain is more tolerant to heat shock than the strain transformed with pPHU231, supporting the involvement of ClpB in rhizobia heat shock tolerance. Both plant growth assays showed that ClpB has an important role in chickpea-rhizobia symbiosis. The nodulation kinetics analysis showed a higher rate of nodule appearance with the clpB-transformed strain. This strain also induced a greater number of nodules and, more notably, its symbiotic effectiveness increased ~60% at pH5 and 83% at pH7, compared to the wild-type strain. Furthermore, a higher frequency of root hair curling was also observed in plants inoculated with the clpB-transformed strain, compared to the wild-type strain. The superior root hair curling induction, nodulation ability and symbiotic effectiveness of the clpB-transformed strain may be explained by an increased expression of symbiosis genes. Indeed, higher transcript levels of the nodulation genes nodA and nodC (~3 folds) were detected in the clpB-transformed strain. The improvement of rhizobia by addition of extra-copies of the clpB gene may be a promising strategy to obtain strains with enhanced stress tolerance and symbiotic effectiveness, thus contributing to their success as crop inoculants, particularly under

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

PubMed

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 (P<0.001) and gender (P=0.034), but not the CTLA-4+49G allele, -318C allele, -318T allele, parental smoking or paternal 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.

Discovering gene annotations in biomedical text databases

PubMed Central

Cakmak, Ali; Ozsoyoglu, Gultekin

2008-01-01

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 pattern occurrences with

You've gotta be lucky: Coverage and the elusive gene-gene interaction.

PubMed

Reimherr, Matthew; Nicolae, Dan L

2011-01-01

Genome-wide association studies (GWAS) have led to a large number of single-SNP association findings, but there has been, so far, no investigation resulting in the discovery of a replicable gene-gene interaction. In this paper, we examine some of the possible explanations for the lack of findings, and argue that coverage of causal variation not only has a large effect on the loss in power, but that the effect is larger than in the single-SNP analyses. We show that the product of linkage disequilibrium measures, r², between causal and tested SNPs offers a good approximation to the loss in efficiency as defined by the ratio of sample sizes that lead to similar power. We also demonstrate that, in addition to the huge search space, the loss in power due to coverage when using commercially available platforms makes the search for gene-gene interactions daunting. © 2010 The Authors Annals of Human Genetics © 2010 Blackwell Publishing Ltd/University College London.

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

PubMed

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.

Integrated Analysis of the Transcriptome and Metabolome of Corynebacterium glutamicum during Penicillin-Induced Glutamic Acid Production.

PubMed

Hirasawa, Takashi; Saito, Masaki; Yoshikawa, Katsunori; Furusawa, Chikara; Shmizu, Hiroshi

2018-05-01

Corynebacterium glutamicum is known for its ability to produce glutamic acid and has been utilized for the fermentative production of various amino acids. Glutamic acid production in C. glutamicum is induced by penicillin. In this study, the transcriptome and metabolome of C. glutamicum is analyzed to understand the mechanism of penicillin-induced glutamic acid production. Transcriptomic analysis with DNA microarray revealed that expression of some glycolysis- and TCA cycle-related genes, which include those encoding the enzymes involved in conversion of glucose to 2-oxoglutaric acid, is upregulated after penicillin addition. Meanwhile, expression of some TCA cycle-related genes, encoding the enzymes for conversion of 2-oxoglutaric acid to oxaloacetic acid, and the anaplerotic reactions decreased. In addition, expression of NCgl1221 and odhI, encoding proteins involved in glutamic acid excretion and inhibition of the 2-oxoglutarate dehydrogenase, respectively, is upregulated. Functional category enrichment analysis of genes upregulated and downregulated after penicillin addition revealed that genes for signal transduction systems are enriched among upregulated genes, whereas those for energy production and carbohydrate and amino acid metabolisms are enriched among the downregulated genes. As for the metabolomic analysis using capillary electrophoresis time-of-flight mass spectrometry, the intracellular content of most metabolites of the glycolysis and the TCA cycle decreased dramatically after penicillin addition. Overall, these results indicate that the cellular metabolism and glutamic acid excretion are mainly optimized at the transcription level during penicillin-induced glutamic acid production by C. glutamicum. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Additional mitochondrial DNA influences the interactions between the nuclear and mitochondrial genomes in a bovine embryo model of nuclear transfer.

PubMed

Srirattana, Kanokwan; St John, Justin C

2018-05-08

We generated cattle embryos using mitochondrial supplementation and somatic cell nuclear transfer (SCNT), named miNT, to determine how additional mitochondrial DNA (mtDNA) modulates the nuclear genome. To eliminate any confounding effects from somatic cell mtDNA in intraspecies SCNT, donor cell mtDNA was depleted prior to embryo production. Additional oocyte mtDNA did not affect embryo development rates but increased mtDNA copy number in blastocyst stage embryos. Moreover, miNT-derived blastocysts had different gene expression profiles when compared with SCNT-derived blastocysts. Additional mtDNA increased expression levels of genes involved in oxidative phosphorylation, cell cycle and DNA repair. Supplementing the embryo culture media with a histone deacetylase inhibitor, Trichostatin A (TSA), had no beneficial effects on the development of miNT-derived embryos, unlike SCNT-derived embryos. When compared with SCNT-derived blastocysts cultured in the presence of TSA, additional mtDNA alone had beneficial effects as the activity of glycolysis may increase and embryonic cell death may decrease. However, these beneficial effects were not found with additional mtDNA and TSA together, suggesting that additional mtDNA alone enhances reprogramming. In conclusion, additional mtDNA increased mtDNA copy number and expression levels of genes involved in energy production and embryo development in blastocyst stage embryos emphasising the importance of nuclear-mitochondrial interactions.

De Novo Assembly, Gene Annotation, and Marker Discovery in Stored-Product Pest Liposcelis entomophila (Enderlein) Using Transcriptome Sequences

PubMed Central

Wei, Dan-Dan; Chen, Er-Hu; Ding, Tian-Bo; Chen, Shi-Chun; Dou, Wei; Wang, Jin-Jun

2013-01-01

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 insecticide resistance

Effect of biochar addition on hydrogen and methane production in two-phase anaerobic digestion of aqueous carbohydrates food waste.

PubMed

Sunyoto, Nimas M S; Zhu, Mingming; Zhang, Zhezi; Zhang, Dongke

2016-11-01

Effect of biochar addition on hydrogen and methane production in two-phase anaerobic digestion of aqueous carbohydrates was studied using bench-scale bioreactors. The cultures with biochar additions were placed in 100ml reactors and incubated at 35°C and pH 5 for hydrogen production. The residual cultures were then used for methane production, incubated at 35°C and pH 7. Daily yields of hydrogen and methane and weekly yield of volatile fatty acids (VFA) were measured. The hydrogen and methane production potentials, rate and lag phases of the two phases were analysed using the Gompertz model. The results showed that biochar addition increased the maximum production rates of hydrogen by 32.5% and methane 41.6%, improved hydrogen yield by 31.0% and methane 10.0%, and shortened the lag phases in the two phases by 36.0% and 41.0%, respectively. Biochar addition also enhanced VFA generation during hydrogen production and VFA degradation in methane production. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

PubMed

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.

Improved polysaccharide production in a submerged culture of Ganoderma lucidum by the heterologous expression of Vitreoscilla hemoglobin gene.

PubMed

Li, Huan-Jun; Zhang, De-Huai; Yue, Tong-Hui; Jiang, Lu-Xi; Yu, Xuya; Zhao, Peng; Li, Tao; Xu, Jun-Wei

2016-01-10

Expression of Vitreoscilla hemoglobin (VHb) gene was used to improve polysaccharide production in Ganoderma lucidum. The VHb gene, vgb, under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase gene promoter was introduced into G. lucidum. The activity of expressed VHb was confirmed by the observation of VHb specific CO-difference spectrum with a maximal absorption at 419 nm for the transformant. The effects of VHb expression on intracellular polysaccharide (IPS) content, extracellular polysaccharide (EPS) production and transcription levels of three genes encoding the enzymes involved in polysaccharide biosynthesis, including phosphoglucomutase (PGM), uridine diphosphate glucose pyrophosphorylase (UGP), and β-1,3-glucan synthase (GLS), were investigated. The maximum IPS content and EPS production in the vgb-bearing G. lucidum were 26.4 mg/100mg dry weight and 0.83 g/L, respectively, which were higher by 30.5% and 88.2% than those of the wild-type strain. The transcription levels of PGM, UGP and GLS were up-regulated by 1.51-, 1.55- and 3.83-fold, respectively, in the vgb-bearing G. lucidum. This work highlights the potential of VHb to enhance G. lucidum polysaccharide production by large scale fermentation. Copyright © 2015 Elsevier B.V. All rights reserved.

Inverse gene-for-gene interactions contribute additively to tan spot susceptibility in wheat

USDA-ARS?s Scientific Manuscript database

Tan spot of wheat, caused by Pyrenophora tritici-repentis, is an important disease in almost all wheat-growing areas of the world. The disease system is known to involve at least three fungal-produced necrotrophic effectors (NEs) that interact with corresponding host sensitivity (S) genes in an inv...

[Modulating expression of key genes within β-carotene synthetic pathway in recombinant Escherichia coli with RBS library to improve β-carotene production].

PubMed

Dai, Guanping; Sun, Tao; Miao, Liangtian; Li, Qingyan; Xiao, Dongguang; Zhang, Xueli

2014-08-01

β-carotene belongs to carotenoids family, widely applied in pharmaceuticals, neutraceuticals, cosmetics and food industries. In this study, three key genes (dxs, idi, and crt operon) within β-carotene synthetic pathway in recombinant Escherichia coli strain CAR005 were modulated with RBS Library to improve β-carotene production. There were 7%, 11% and 17% increase of β-carotene yield respectively after modulating dxs, idi and crt operon genes with RBS Library, demonstrating that modulating gene expression with regulatory parts libraries would have more opportunities to obtain optimal production of target compound. Combined modulation of crt operon, dxs and idi genes led to 35% increase of β-carotene yield compared to parent strain CAR005. The optimal gene expression strength identified in single gene modulation would not be the optimal strength when used in combined modulation. Our study provides a new strategy for improving production of target compound through modulation of gene expression.

Guaranteeing a captive audience: coordinated regulation of gene transfer agent (GTA) production and recipient capability by cellular regulators.

PubMed

Westbye, Alexander B; Beatty, J Thomas; Lang, Andrew S

2017-08-01

Gene transfer agents (GTAs) are bacteriophage-like particles produced by many prokaryotes. Several members of the Alphaproteobacteria produce a class of genetically-related GTAs that is best studied in Rhodobacter capsulatus. DNA transfer by the R. capsulatus GTA (RcGTA) combines aspects of both transduction and natural transformation, as recipient cells require a natural transformation-like system to incorporate donated DNA. The genes involved in RcGTA production and recipient capability are located at multiple loci in the bacterial genome; however, a conserved phosphorelay containing the response regulator CtrA and a quorum sensing system regulate both RcGTA production and recipient capability. This review highlights recent discoveries in RcGTA biology, and focuses on the co-regulation of genes involved in RcGTA production and recipient capability. Copyright © 2017 Elsevier Ltd. All rights reserved.

Improving aerobic stability and biogas production of maize silage using silage additives.

PubMed

Herrmann, Christiane; Idler, Christine; Heiermann, Monika

2015-12-01

The effects of air stress during storage, exposure to air at feed-out, and treatment with silage additives to enhance aerobic stability on methane production from maize silage were investigated at laboratory scale. Up to 17% of the methane potential of maize without additive was lost during seven days exposure to air on feed-out. Air stress during storage reduced aerobic stability and further increased methane losses. A chemical additive containing salts of benzoate and propionate, and inoculants containing heterofermentative lactic acid bacteria were effective to increase aerobic stability and resulted in up to 29% higher methane yields after exposure to air. Exclusion of air to the best possible extent and high aerobic stabilities should be primary objectives when ensiling biogas feedstocks. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

[Cloning of Enterobacter aerogenes fh1A gene and overexpression of hydrogen production].

PubMed

Zhao, Jinfang; Song, Wenlu; Cheng, Jun; Zhang, Chuanxi

2010-06-01

We amplified and overexpressed the FHL activator (fh1A) in E. aerogenes ATCC13408 to enhance hydrogen production. By using universal primers and genome walking, we cloned the full open reading frame (ORF) of fh1A gene. We inserted it into the glutathion S-transferase (GST) fusion expression vector pGEX4T-2-Cat, and transformed the recombinant plasmid into E. aerogenes ATCC13408 via electroporation for expression. Then we measured the hydrogen production of the recombinant strain in a batch culture. We found that the ORF of fh1A was 2073 base pair in length, potential to encode a 690 amino acid peptide (GenBank accession GU188474). The Fh1A protein from E. aerogenes ATCC13408 shared high amino acid identities with those from other bacterial species. By using SDS-PAGE and Western blot analysis, we confirmed that the fh1A gene had successfully expressed in the strain. The hydrogen yield of the recombinant strain was increased from 1.23 to 1.48 mol H2/mol glucose. [ Conclusion ] Enhancement of hydrogen productivity was attained under anaerobic conditions with the recombinant strain.

Characterization of the Expression of the Petunia Glycine-Rich Protein-1 Gene Product 1

PubMed Central

Condit, Carol M.; McLean, B. Gail; Meagher, Richard B.

1990-01-01

We have examined the expression of the petunia (Petunia hybrida) glycine-rich protein-1 (ptGRP1) gene product using an antibody raised against a synthetic peptide comprising amino acids 22 through 36 of the mature ptGRP1 protein. This antibody recognizes a single protein of 23 kilodaltons. Cell fractionation studies showed that, as predicted (CM Condit, RB Meagher [1986] Nature 323: 178-181), ptGRP1 is most likely localized in the cell wall. In addition, it was found that (extractable) ptGRP1 is present in much higher abundance in unexpanded than in fully expanded tissue, with highest levels of accumulation in the bud. This same developmentally regulated pattern of protein expression was found in all varieties of petunia tested. In addition, tissue blots of petunia stem sections showed that ptGRP1 is localized to within the vascular tissue (to at least the phloem or cambium) and to either the epidermal cells or to a layer of collenchyma cells directly below the epidermis. Localization of ptGRP1 antigen in these cell types is shown to occur at different times in the overall development of the plant and at different quantitative levels. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:16667509

Control of aflatoxin production of Aspergillus flavus and Aspergillus parasiticus using RNA silencing technology by targeting aflD (nor-1) gene.

PubMed

Abdel-Hadi, Ahmed M; Caley, Daniel P; Carter, David R F; Magan, Naresh

2011-06-01

Aspergillus flavus and Aspergillus parasiticus are important pathogens of cotton, corn, peanuts and other oil-seed crops, producing toxins both in the field and during storage. We have designed three siRNA sequences (Nor-Ia, Nor-Ib, Nor-Ic) to target the mRNA sequence of the aflD gene to examine the potential for using RNA silencing technology to control aflatoxin production. Thus, the effect of siRNAs targeting of two key genes in the aflatoxin biosynthetic pathway, aflD (structural) and aflR (regulatory gene) and on aflatoxin B(1 )(AFB(1)), and aflatoxin G(1) (AFG(1)) production was examined. The study showed that Nor-Ib gave a significant decrease in aflD mRNA, aflR mRNA abundance, and AFB(1) production (98, 97 and 97% when compared to the controls) in A. flavus NRRL3357, respectively. Reduction in aflD and aflR mRNA abundance and AFB(1 )production increased with concentration of siRNA tested. There was a significant inhibition in aflD and AFB(1) production by A. flavus EGP9 and AFG(1 )production by A. parasiticus NRRL 13005. However, there was no significant decrease in AFG(1) production by A. parasiticus SSWT 2999. Changes in AFB(1) production in relation to mRNA levels of aflD showed a good correlation (R = 0.88; P = 0.00001); changes in aflR mRNA level in relation to mRNA level of aflD also showed good correlation (R = 0.82; P = 0.0001). The correlations between changes in aflR and aflD gene expression suggests a strong relationship between these structural and regulatory genes, and that aflD could be used as a target gene to develop efficient means for aflatoxin control using RNA silencing technology.

Deletion of acetate transporter gene ADY2 improved tolerance of Saccharomyces cerevisiae against multiple stresses and enhanced ethanol production in the presence of acetic acid.

PubMed

Zhang, Mingming; Zhang, Keyu; Mehmood, Muhammad Aamer; Zhao, Zongbao Kent; Bai, Fengwu; Zhao, Xinqing

2017-12-01

The aim of this work was to study the effects of deleting acetate transporter gene ADY2 on growth and fermentation of Saccharomyces cerevisiae in the presence of inhibitors. Comparative transcriptome analysis revealed that three genes encoding plasma membrane carboxylic acid transporters, especially ADY2, were significantly downregulated under the zinc sulfate addition condition in the presence of acetic acid stress, and the deletion of ADY2 improved growth of S. cerevisiae under acetic acid, ethanol and hydrogen peroxide stresses. Consistently, a concomitant increase in ethanol production by 14.7% in the presence of 3.6g/L acetic acid was observed in the ADY2 deletion mutant of S. cerevisiae BY4741. Decreased intracellular acetic acid, ROS accumulation, and plasma membrane permeability were observed in the ADY2 deletion mutant. These findings would be useful for developing robust yeast strains for efficient ethanol production. Copyright © 2017 Elsevier Ltd. All rights reserved.

Germline mutations in candidate predisposition genes in individuals with cutaneous melanoma and at least two independent additional primary cancers.

PubMed

Pritchard, Antonia L; Johansson, Peter A; Nathan, Vaishnavi; Howlie, Madeleine; Symmons, Judith; Palmer, Jane M; Hayward, Nicholas K

2018-01-01

While a number of autosomal dominant and autosomal recessive cancer syndromes have an associated spectrum of cancers, the prevalence and variety of cancer predisposition mutations in patients with multiple primary cancers have not been extensively investigated. An understanding of the variants predisposing to more than one cancer type could improve patient care, including screening and genetic counselling, as well as advancing the understanding of tumour development. A cohort of 57 patients ascertained due to their cutaneous melanoma (CM) diagnosis and with a history of two or more additional non-cutaneous independent primary cancer types were recruited for this study. Patient blood samples were assessed by whole exome or whole genome sequencing. We focussed on variants in 525 pre-selected genes, including 65 autosomal dominant and 31 autosomal recessive cancer predisposition genes, 116 genes involved in the DNA repair pathway, and 313 commonly somatically mutated in cancer. The same genes were analysed in exome sequence data from 1358 control individuals collected as part of non-cancer studies (UK10K). The identified variants were classified for pathogenicity using online databases, literature and in silico prediction tools. No known pathogenic autosomal dominant or previously described compound heterozygous mutations in autosomal recessive genes were observed in the multiple cancer cohort. Variants typically found somatically in haematological malignancies (in JAK1, JAK2, SF3B1, SRSF2, TET2 and TYK2) were present in lymphocyte DNA of patients with multiple primary cancers, all of whom had a history of haematological malignancy and cutaneous melanoma, as well as colorectal cancer and/or prostate cancer. Other potentially pathogenic variants were discovered in BUB1B, POLE2, ROS1 and DNMT3A. Compared to controls, multiple cancer cases had significantly more likely damaging mutations (nonsense, frameshift ins/del) in tumour suppressor and tyrosine kinase genes and

Evolution of Gene Duplication in Plants.

PubMed

Panchy, Nicholas; Lehti-Shiu, Melissa; Shiu, Shin-Han

2016-08-01

Ancient duplication events and a high rate of retention of extant pairs of duplicate genes have contributed to an abundance of duplicate genes in plant genomes. These duplicates have contributed to the evolution of novel functions, such as the production of floral structures, induction of disease resistance, and adaptation to stress. Additionally, recent whole-genome duplications that have occurred in the lineages of several domesticated crop species, including wheat (Triticum aestivum), cotton (Gossypium hirsutum), and soybean (Glycine max), have contributed to important agronomic traits, such as grain quality, fruit shape, and flowering time. Therefore, understanding the mechanisms and impacts of gene duplication will be important to future studies of plants in general and of agronomically important crops in particular. In this review, we survey the current knowledge about gene duplication, including gene duplication mechanisms, the potential fates of duplicate genes, models explaining duplicate gene retention, the properties that distinguish duplicate from singleton genes, and the evolutionary impact of gene duplication. © 2016 American Society of Plant Biologists. All Rights Reserved.

Type II thioesterase gene (ECO-orf27) from Amycolatopsis orientalis influences production of the polyketide antibiotic, ECO-0501 (LW01).

PubMed

Shen, Yang; Huang, He; Zhu, Li; Luo, Minyu; Chen, Daijie

2012-11-01

ECO-orf27 associated with the cluster of ECO-0501 (LW01) from Amycolatopsis orientalis is deduced to encode a type II thioesterase. Disruption of ECO-orf27 reduced LW01 production by 95 %. Complementation of the disrupted mutant with intact ECO-orf27 restored the production of LW01 suggesting that ECO-orf27 is crucial for LW01 biosynthesis. ECO-TE I, the gene encoding type I thioesterase from LW01 polyketide synthases, cannot complement ECO-orf27 deficient mutant distinguishing ECO-orf27 from type I thioesterase gene. Type II thioesterase gene pikAV from Streptomyces venezuelae could complement ECO-orf27 in A. orientalis indicating that the two genes are equivalent in their function. Overexpression of ECO-orf27 resulted in a 20 % increase in LW01 production providing an alternative approach for yield improvement.

Production of Null Mutants in the Major Intestinal Esterase Gene (Ges-1) of the Nematode Caenorhabditis Elegans

PubMed Central

McGhee, J. D.; Birchall, J. C.; Chung, M. A.; Cottrell, D. A.; Edgar, L. G.; Svendsen, P. C.; Ferrari, D. C.

1990-01-01

The ges-1 gene of the nematode Caenorhabditis elegans codes for a nonspecific carboxylesterase that is expressed only in the intestinal lineage. This esterase has turned out to be a convenient biochemical marker for lineage-specific differentiation. In the present paper, we describe the production of several C. elegans strains that lack detectable activity of the ges-1 esterase. To isolate these ges-1 null strains, we first produced a strain of hermaphrodites in which the wild-type copy of the ges-1 gene was stably balanced over a previously isolated isoelectric focusing allele, ges-1(ca6); this parental strain was then mutagenized with EMS and isoelectric focusing gels were used to identify progeny populations that lacked either ges-1(+) or ges-1(ca6) esterase activity. This method is a straightforward and general approach to obtaining null mutations in any gene that has a biochemical or immunological assay. The ges-1 gene is not essential to worm survival, development or reproduction. Furthermore, lack of the ges-1 product has no obvious effect on the ability of worms (containing either normal or greatly reduced levels of acetylcholinesterases) to survive exposure to esterase inhibitors. The ges-1 gene product provides roughly half of the total esterase activity measured in crude extracts of L1 larvae or mixed worm populations. However, histochemical staining of individual ges-1(0) embryos shows that the ges-1 esterase is the first and essentially the only esterase to be produced during embryonic development, from the midproliferation phase up to at least the twofold stage of morphogenesis. These ges-1(0) strains now allow us to investigate the developmental control of the ges-1 gene by DNA-mediated transformation, in which the ges-1 gene acts as its own reporter. PMID:2379823

Nonsteroidal anti-inflammatory drug activated gene-1 (NAG-1) modulators from natural products as anti-cancer agents

USDA-ARS?s Scientific Manuscript database

Natural products are rich source of gene modulators for prevention and treatment of cancer. In recent days, nonsteroidal anti-inflammatory drug (NSAID) activated gene-1 (NAG-1) has been focused as a new target of diverse cancers like colorectal, pancreatic, prostate, and breast. A variety of natural...

Effect of red mud addition on tetracycline and copper resistance genes and microbial community during the full scale swine manure composting.

PubMed

Wang, Rui; Zhang, Junya; Sui, Qianwen; Wan, Hefeng; Tong, Juan; Chen, Meixue; Wei, Yuansong; Wei, Dongbin

2016-09-01

Swine manure has been considered as the reservoir of antibiotic resistance genes (ARGs). Composting is one of the most suitable technologies for treating livestock manures, and red mud was proved to have a positive effect on nitrogen conservation during composting. This study investigated the abundance of eight tetracycline and three copper resistance genes, the bacterial community during the full scale swine manure composting with or without addition of red mud. The results showed that ARGs in swine manure could be effectively removed through composting (reduced by 2.4log copies/g TS), especially during the thermophilic phase (reduced by 1.5log copies/g TS), which the main contributor might be temperature. Additionally, evolution of bacterial community could also have a great influence on ARGs. Although addition of red mud could enhance nitrogen conservation, it obviously hindered removal of ARGs (reduced by 1.7log copies/g TS) and affected shaping of bacterial community during composting. Copyright © 2016 Elsevier Ltd. All rights reserved.

Association between a polymorphism of the α-lactalbumin gene and milk production traits in Chinese Holstein cows.

PubMed

Zhou, J P; Dong, C H

2013-09-04

The traits particularly important for milk production include milk yield, protein percentage, fat percentage, and the somatic cell score. Alpha-lactalbumin (α-LA) is an important whey protein of cow milk, and is also present in the milk of many other mammalian species. In this study, we analyzed the genetic polymorphisms of the α-LA gene and their relationship to milk production traits (milk yield, protein percentage, fat percentage, and somatic cell score) in Chinese Holstein cows. The goal of this study was to contribute further molecular genetic information related to dairy cattle, to determine the molecular markers that are most closely linked with milk production traits, and to provide a scientific basis for the improvement of economically relevant traits in cows. Fluorescence-based conformation-sensitive gel electrophoresis, DNA sequencing, and ligation detection reaction techniques were used to analyze genetic variations of the α-LA gene (5'-UTR, exons 1, 2, 3, 4, and 3'-UTR) in 923 Chinese Holstein cows. One novel single nucleotide polymorphism (SNP), α-LA2516, was identified in exon 4 of the α-LA gene. Allele frequencies were as follows: T 0.674, C 0.326. Association analysis revealed that α-LA2516 was not associated with milk yield, protein percentage, fat percentage, or somatic cell score (P > 0.05). These findings suggest that the SNP α-LA2516 in the α-LA gene likely does not have potential as a molecular marker for milk production traits in Chinese Holstein cows.

Functional assessment of plant and microalgal lipid pathway genes in yeast to enhance microbial industrial oil production.

PubMed

Peng, Huadong; Moghaddam, Lalehvash; Brinin, Anthony; Williams, Brett; Mundree, Sagadevan; Haritos, Victoria S

2018-03-01

As promising alternatives to fossil-derived oils, microbial lipids are important as industrial feedstocks for biofuels and oleochemicals. Our broad aim is to increase lipid content in oleaginous yeast through expression of lipid accumulation genes and use Saccharomyces cerevisiae to functionally assess genes obtained from oil-producing plants and microalgae. Lipid accumulation genes DGAT (diacylglycerol acyltransferase), PDAT (phospholipid: diacylglycerol acyltransferase), and ROD1 (phosphatidylcholine: diacylglycerol choline-phosphotransferase) were separately expressed in yeast and lipid production measured by fluorescence, solvent extraction, thin layer chromatography, and gas chromatography (GC) of fatty acid methyl esters. Expression of DGAT1 from Arabidopsis thaliana effectively increased total fatty acids by 1.81-fold above control, and ROD1 led to increased unsaturated fatty acid content of yeast lipid. The functional assessment approach enabled the fast selection of candidate genes for metabolic engineering of yeast for production of lipid feedstocks. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

Deletion of the hfsB gene increases ethanol production in Thermoanaerobacterium saccharolyticum and several other thermophilic anaerobic bacteria

DOE PAGES

Eminoglu, Aysenur; Murphy, Sean Jean-Loup; Maloney, Marybeth; ...

2017-11-30

With the discovery of interspecies hydrogen transfer in the late 1960s (Bryant et al. in Arch Microbiol 59:20–31, 1967), it was shown that reducing the partial pressure of hydrogen could cause mixed acid fermenting organisms to produce acetate at the expense of ethanol. Hydrogen and ethanol are both more reduced than glucose. Thus there is a tradeoff between production of these compounds imposed by electron balancing requirements; however, the mechanism is not fully known. Deletion of the hfsA or B subunits resulted in a roughly 1.8-fold increase in ethanol yield. The increase in ethanol production appears to be associated withmore » an increase in alcohol dehydrogenase activity, which appears to be due, at least in part, to increased expression of the adhE gene, and may suggest a regulatory linkage between hfsB and adhE. We studied this system most intensively in the organism Thermoanaerobacterium saccharolyticum; however, deletion of hfsB also increases ethanol production in other thermophilic bacteria suggesting that this could be used as a general technique for engineering thermophilic bacteria for improved ethanol production in organisms with hfs-type hydrogenases. Since its discovery by Shaw et al. (JAMA 191:6457–64, 2009), the hfs hydrogenase has been suspected to act as a regulator due to the presence of a PAS domain. We provide additional support for the presence of a regulatory phenomenon. In addition, we find a practical application for this scientific insight, namely increasing ethanol yield in strains that are of interest for ethanol production from cellulose or hemicellulose. In two of these organisms (T. xylanolyticum and T. thermosaccharolyticum), the ethanol yields are the highest reported to date.« less

Deletion of the hfsB gene increases ethanol production in Thermoanaerobacterium saccharolyticum and several other thermophilic anaerobic bacteria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eminoglu, Aysenur; Murphy, Sean Jean-Loup; Maloney, Marybeth

With the discovery of interspecies hydrogen transfer in the late 1960s (Bryant et al. in Arch Microbiol 59:20–31, 1967), it was shown that reducing the partial pressure of hydrogen could cause mixed acid fermenting organisms to produce acetate at the expense of ethanol. Hydrogen and ethanol are both more reduced than glucose. Thus there is a tradeoff between production of these compounds imposed by electron balancing requirements; however, the mechanism is not fully known. Deletion of the hfsA or B subunits resulted in a roughly 1.8-fold increase in ethanol yield. The increase in ethanol production appears to be associated withmore » an increase in alcohol dehydrogenase activity, which appears to be due, at least in part, to increased expression of the adhE gene, and may suggest a regulatory linkage between hfsB and adhE. We studied this system most intensively in the organism Thermoanaerobacterium saccharolyticum; however, deletion of hfsB also increases ethanol production in other thermophilic bacteria suggesting that this could be used as a general technique for engineering thermophilic bacteria for improved ethanol production in organisms with hfs-type hydrogenases. Since its discovery by Shaw et al. (JAMA 191:6457–64, 2009), the hfs hydrogenase has been suspected to act as a regulator due to the presence of a PAS domain. We provide additional support for the presence of a regulatory phenomenon. In addition, we find a practical application for this scientific insight, namely increasing ethanol yield in strains that are of interest for ethanol production from cellulose or hemicellulose. In two of these organisms (T. xylanolyticum and T. thermosaccharolyticum), the ethanol yields are the highest reported to date.« less

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

PubMed

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tamano, Koichi; Bruno, Kenneth S.; Karagiosis, Sue A.

2013-01-01

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

Crystallization and preliminary X-ray analysis of gene product 44 from bacteriophage Mu

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kondou, Youhei; Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Toyama; Kitazawa, Daisuke

2005-01-01

Bacteriophage Mu baseplate protein gene product 44 was crystallized. The crystal belongs to space group R3, with unit-cell parameters a = b = 126.6, c = 64.2 Å. Bacteriophage Mu baseplate protein gene product 44 (gp44) is an essential protein required for the assembly of viable phages. To investigate the roles of gp44 in baseplate assembly and infection, gp44 was crystallized at pH 6.0 in the presence of 20% 2-methyl-2,4-pentanediol. The crystals belong to space group R3, with unit-cell parameters a = b = 127.47, c = 63.97 Å. The crystals diffract X-rays to at least 2.1 Å resolution andmore » are stable in the X-ray beam and are therefore appropriate for structure determination. Native data have been collected to 2.1 Å resolution using a DIP6040 image-plate system at beamline BL44XU at the SPring-8 facility in Japan.« less

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

PubMed

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.

Distribution and Genetic Diversity of Bacteriocin Gene Clusters in Rumen Microbial Genomes.

PubMed

Azevedo, Analice C; Bento, Cláudia B P; Ruiz, Jeronimo C; Queiroz, Marisa V; Mantovani, Hilário C

2015-10-01

Some species of ruminal bacteria are known to produce antimicrobial peptides, but the screening procedures have mostly been based on in vitro assays using standardized methods. Recent sequencing efforts have made available the genome sequences of hundreds of ruminal microorganisms. In this work, we performed genome mining of the complete and partial genome sequences of 224 ruminal bacteria and 5 ruminal archaea to determine the distribution and diversity of bacteriocin gene clusters. A total of 46 bacteriocin gene clusters were identified in 33 strains of ruminal bacteria. Twenty gene clusters were related to lanthipeptide biosynthesis, while 11 gene clusters were associated with sactipeptide production, 7 gene clusters were associated with class II bacteriocin production, and 8 gene clusters were associated with class III bacteriocin production. The frequency of strains whose genomes encode putative antimicrobial peptide precursors was 14.4%. Clusters related to the production of sactipeptides were identified for the first time among ruminal bacteria. BLAST analysis indicated that the majority of the gene clusters (88%) encoding putative lanthipeptides contained all the essential genes required for lanthipeptide biosynthesis. Most strains of Streptococcus (66.6%) harbored complete lanthipeptide gene clusters, in addition to an open reading frame encoding a putative class II bacteriocin. Albusin B-like proteins were found in 100% of the Ruminococcus albus strains screened in this study. The in silico analysis provided evidence of novel biosynthetic gene clusters in bacterial species not previously related to bacteriocin production, suggesting that the rumen microbiota represents an underexplored source of antimicrobial peptides. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Reciprocal Translocation Observed in End-of-Production Cells of a Commercial CHO-Based Process.

PubMed

Rouiller, Yolande; Kleuser, Beate; Toso, Emiliano; Palinksy, Wolf; Rossi, Mara; Rossatto, Paola; Barberio, Davide; Broly, Hervé

2015-01-01

During the validation of an additional working cell bank derived from a validated master cell bank to support the commercial production continuum of a recombinant protein, we observed an unexpected chromosomal location of the gene of interest in some end-of-production cells. This event-identified by fluorescence in situ hybridization and multicolour chromosome painting as a reciprocal translocation involving a chromosome region containing the gene of interest with its integral coding and flanking sequences-was unique, occurred probably during or prior to multicolour chromosome painting establishment, and was transmitted to the descending generations. Cells bearing the translocation had a transient and process-independent selective advantage, which did not affect process performance and product quality. However, this first report of a translocation affecting the gene of interest location in Chinese Hamster Ovary cells used for producing a biotherapeutic indicates the importance of the demonstration of the integrity of the gene of interest in end-of-production cells. The expression of recombinant therapeutic proteins in mammalian cells depends on the establishment of a cell line with the gene of interest integrated in the host genome and stably expressed over time. Before being used for commercial production, cell lines are submitted to a qualification program in order to ensure their phenotypic and genotypic characteristics and the efficacy and safety of the product. During the production life cycle of a therapeutic protein, additional cells banks have to be validated after exhaustion of the current qualified cell bank in order to support the commercial production continuum of the recombinant protein. It is during the validation of an additional working cell bank derived from a validated master cell bank that we detected a different chromosome bearing the gene of interest in a portion of cells at the end of the upstream production phase. In our case, this event did

Seagrass metabolism across a productivity gradient using the eddy covariance, Eulerian control volume, and biomass addition techniques

NASA Astrophysics Data System (ADS)

Long, Matthew H.; Berg, Peter; Falter, James L.

2015-05-01

The net ecosystem metabolism of the seagrass Thalassia testudinum was studied across a nutrient and productivity gradient in Florida Bay, Florida, using the Eulerian control volume, eddy covariance, and biomass addition techniques. In situ oxygen fluxes were determined by a triangular Eulerian control volume with sides 250 m long and by eddy covariance instrumentation at its center. The biomass addition technique evaluated the aboveground seagrass productivity through the net biomass added. The spatial and temporal resolutions, accuracies, and applicability of each method were compared. The eddy covariance technique better resolved the short-term flux rates and the productivity gradient across the bay, which was consistent with the long-term measurements from the biomass addition technique. The net primary production rates from the biomass addition technique, which were expected to show greater autotrophy due to the exclusion of sediment metabolism and belowground production, were 71, 53, and 30 mmol carbon m-2 d-1 at 3 sites across the bay. The net ecosystem metabolism was 35, 25, and 11 mmol oxygen m-2 d-1 from the eddy covariance technique and 10, -103, and 14 mmol oxygen m-2 d-1 from the Eulerian control volume across the same sites, respectively. The low-flow conditions in the shallow bays allowed for periodic stratification and long residence times within the Eulerian control volume that likely reduced its precision. Overall, the eddy covariance technique had the highest temporal resolution while producing accurate long-term flux rates that surpassed the capabilities of the biomass addition and Eulerian control volume techniques in these shallow coastal bays.

Discovery of a Phosphonoacetic Acid Derived Natural Product by Pathway Refactoring.

PubMed

Freestone, Todd S; Ju, Kou-San; Wang, Bin; Zhao, Huimin

2017-02-17

The activation of silent natural product gene clusters is a synthetic biology problem of great interest. As the rate at which gene clusters are identified outpaces the discovery rate of new molecules, this unknown chemical space is rapidly growing, as too are the rewards for developing technologies to exploit it. One class of natural products that has been underrepresented is phosphonic acids, which have important medical and agricultural uses. Hundreds of phosphonic acid biosynthetic gene clusters have been identified encoding for unknown molecules. Although methods exist to elicit secondary metabolite gene clusters in native hosts, they require the strain to be amenable to genetic manipulation. One method to circumvent this is pathway refactoring, which we implemented in an effort to discover new phosphonic acids from a gene cluster from Streptomyces sp. strain NRRL F-525. By reengineering this cluster for expression in the production host Streptomyces lividans, utility of refactoring is demonstrated with the isolation of a novel phosphonic acid, O-phosphonoacetic acid serine, and the characterization of its biosynthesis. In addition, a new biosynthetic branch point is identified with a phosphonoacetaldehyde dehydrogenase, which was used to identify additional phosphonic acid gene clusters that share phosphonoacetic acid as an intermediate.

Indications of Transformation Products from Hydraulic Fracturing Additives in Shale Gas Wastewater

NASA Astrophysics Data System (ADS)

Elsner, Martin; Hoelzer, Kathrin; Sumner, Andrew J.; Karatum, Osman; Nelson, Robert K.; Drollette, Brian D.; O'Connor, Megan P.; D'Ambro, Emma; Getzinger, Gordon J.; Ferguson, P. Lee; Reddy, Christopher M.; Plata, Desiree L.

2016-04-01

Unconventional natural gas development (UNGD) generates large volumes of wastewater, whose detailed composition must be known for adequate risk assessment and treatment. In particular, there is a need to elucidate the structures of organic chemical additives, extracted geogenic compounds, and transformation products. This study investigated six Fayetteville Shale UNGD wastewater samples for their organic composition using purge-and-trap gas chromatography-mass spectrometry (P&T-GC-MS) in combination with liquid-liquid extraction with comprehensive two-dimensional gas chromatography-time of flight-mass spectrometry (GCxGC-TOF-MS). Following application of strict compound identification confidence criteria, we classified compounds according to their putative origin. Samples displayed distinct chemical distributions composed of typical geogenic substances (hydrocarbons), disclosed UNGD additives (e.g., hydrocarbons, phthalates, such as diisobutyl phthalate, and radical initiators, such as azobisisobutyronitrile), and undisclosed compounds (e.g., halogenated hydrocarbons, such as 2-bromohexane or 4-bromoheptane). Undisclosed chloromethyl alkanoates (chloromethyl propanoate, pentanoate, and octanoate) were identified as putative delayed acids (those that release acidic moieties only after hydrolytic cleavage, whose rate could potentially be controlled), suggesting they were deliberately introduced to react in the subsurface. Identification of halogenated methanes and acetones, in contrast, suggested they were formed as unintended by-products. Our study highlights the possibility that UNGD operations generate transformation products, knowledge of which is crucial for risk assessment and treatment strategies, and underscores the value of disclosing potential precursors that are injected into the subsurface.

Homologous gene replacement in Physarum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Burland, T.G.; Pallotta, D.

1995-01-01

The protist Physarum polycephalum is useful for analysis of several aspects of cellular and developmental biology. To expand the opportunities for experimental analysis of this organism, we have developed a method for gene replacement. We transformed Physarum amoebae with plasmid DNA carrying a mutant allele, ardD{Delta}1, of the ardD actin gene; ardD{Delta}1 mutates the critical carboxy-terminal region of the gene product. Because ardD is not expressed in the amoeba, replacement of ardD{sup +} with ardD{Delta}1 should not be lethal for this cell type. Transformants were obtained only when linear plasmid DNA was used. Most transformants carried one copy of ardD{Delta}1more » in addition to ardD{sup +}, but in two (5%), ardD{sup +} was replaced by a single copy of ardD{Delta}1. This is the first example of homologous gene replacement in Physarum. ardD{Delta}1 was stably maintained in the genome through growth, development and meiosis. We found no effect of ardD{Delta}l on viability, growth, or development of any of the various cell types of Physarum. Thus, the carboxy-terminal region of the ardD product appears not to perform a unique essential role in growth or development. Nevertheless, this method for homologous gene replacement can be applied to analyze the function of any cloned gene. 38 refs., 6 figs., 1 tab.« less

ALOX5 gene variants affect eicosanoid production and response to fish oil supplementation

USDA-ARS?s Scientific Manuscript database

The objective of this study was to determine whether 5-lipoxygenase (ALOX5) gene variants associated with cardiovascular disease affect eicosanoid production by monocytes. The study was a randomized, double-masked, parallel intervention trial with fish oil (5.0 g of fish oil daily, containing 2.0 g ...

Computational gene network study on antibiotic resistance genes of Acinetobacter baumannii.

PubMed

Anitha, P; Anbarasu, Anand; Ramaiah, Sudha

2014-05-01

Multi Drug Resistance (MDR) in Acinetobacter baumannii is one of the major threats for emerging nosocomial infections in hospital environment. Multidrug-resistance in A. baumannii may be due to the implementation of multi-combination resistance mechanisms such as β-lactamase synthesis, Penicillin-Binding Proteins (PBPs) changes, alteration in porin proteins and in efflux pumps against various existing classes of antibiotics. Multiple antibiotic resistance genes are involved in MDR. These resistance genes are transferred through plasmids, which are responsible for the dissemination of antibiotic resistance among Acinetobacter spp. In addition, these resistance genes may also have a tendency to interact with each other or with their gene products. Therefore, it becomes necessary to understand the impact of these interactions in antibiotic resistance mechanism. Hence, our study focuses on protein and gene network analysis on various resistance genes, to elucidate the role of the interacting proteins and to study their functional contribution towards antibiotic resistance. From the search tool for the retrieval of interacting gene/protein (STRING), a total of 168 functional partners for 15 resistance genes were extracted based on the confidence scoring system. The network study was then followed up with functional clustering of associated partners using molecular complex detection (MCODE). Later, we selected eight efficient clusters based on score. Interestingly, the associated protein we identified from the network possessed greater functional similarity with known resistance genes. This network-based approach on resistance genes of A. baumannii could help in identifying new genes/proteins and provide clues on their association in antibiotic resistance. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

NASA Astrophysics Data System (ADS)

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.

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

PubMed

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

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-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.

Predicting Gene Structures from Multiple RT-PCR Tests

NASA Astrophysics Data System (ADS)

Kováč, Jakub; Vinař, Tomáš; Brejová, Broňa

It has been demonstrated that the use of additional information such as ESTs and protein homology can significantly improve accuracy of gene prediction. However, many sources of external information are still being omitted from consideration. Here, we investigate the use of product lengths from RT-PCR experiments in gene finding. We present hardness results and practical algorithms for several variants of the problem and apply our methods to a real RT-PCR data set in the Drosophila genome. We conclude that the use of RT-PCR data can improve the sensitivity of gene prediction and locate novel splicing variants.

Improvement of L-valine production at high temperature in Brevibacterium flavum by overexpressing ilvEBNrC genes.

PubMed

Hou, Xiaohu; Ge, Xiangyang; Wu, Di; Qian, He; Zhang, Weiguo

2012-01-01

Brevibacterium flavum ATCC14067 was engineered for L: -valine production by overexpression of different ilv genes; the ilvEBN(r)C genes from B. flavum NV128 provided the best candidate for L: -valine production. In traditional fermentation, L: -valine production reached 30.08 ± 0.92 g/L at 31°C in 72 h with a low conversion efficiency of 0.129 g/g. To further improve the L: -valine production and conversion efficiency based on the optimum temperatures of L: -valine biosynthesis enzymes (above 35°C) and the thermotolerance of B. flavum, the fermentation temperature was increased to 34, 37, and 40°C. As a result, higher metabolic rate and L: -valine biosynthesis enzymes activity were obtained at high temperature, and the maximum L: -valine production, conversion efficiency, and specific L: -valine production rate reached 38.08 ± 1.32 g/L, 0.241 g/g, and 0.133 g g(-1) h(-1), respectively, at 37°C in 48 h fermentation. The strategy for enhancing L: -valine production by overexpression of key enzymes in thermotolerant strains may provide an alternative approach to enhance branched-chain amino acids production with other strains.

Improvement of the ammonia assimilation for enhancing L-arginine production of Corynebacterium crenatum.

PubMed

Guo, Jing; Man, Zaiwei; Rao, Zhiming; Xu, Meijuan; Yang, Taowei; Zhang, Xian; Xu, Zhenghong

2017-03-01

There are four nitrogen atoms in L-arginine molecule and the nitrogen content is 32.1%. By now, metabolic engineering for L-arginine production strain improvement was focused on carbon flux optimization. In previous work, we obtained an L-arginine-producing Corynebacterium crenatum SDNN403 (ARG) through screening and mutation breeding. In this paper, a strain engineering strategy focusing on nitrogen supply and ammonium assimilation for L-arginine production was performed. Firstly, the effects of nitrogen atom donor (L-glutamate, L-glutamine and L-aspartate) addition on L-arginine production of ARG were studied, and the addition of L-glutamine and L-aspartate was beneficial for L-arginine production. Then, the glutamine synthetase gene glnA and aspartase gene aspA from E. coli were overexpressed in ARG for increasing the L-glutamine and L-aspartate synthesis, and the L-arginine production was effectively increased. In addition, the L-glutamate supply re-emerged as a limiting factor for L-arginine biosynthesis. Finally, the glutamate dehydrogenase gene gdh was co-overexpressed for further enhancement of L-arginine production. The final strain could produce 53.2 g l -1 of L-arginine, which was increased by 41.5% compared to ARG in fed-batch fermentation.

Dropwise additive manufacturing of pharmaceutical products for amorphous and self emulsifying drug delivery systems.

PubMed

Içten, Elçin; Purohit, Hitesh S; Wallace, Chelsey; Giridhar, Arun; Taylor, Lynne S; Nagy, Zoltan K; Reklaitis, Gintaras V

2017-05-30

The improvements in healthcare systems and the advent of the precision medicine initiative have created the need to develop more innovative manufacturing methods for the delivery and production of individualized dosing and personalized treatments. In accordance with the changes observed in healthcare systems towards more innovative therapies, this paper presents dropwise additive manufacturing of pharmaceutical products (DAMPP) for small scale, distributed manufacturing of individualized dosing as an alternative to conventional manufacturing methods A dropwise additive manufacturing process for amorphous and self-emulsifying drug delivery systems is reported, which utilizes drop-on-demand printing technology for automated and controlled deposition of melt-based formulations onto inert tablets. The advantages of drop on demand technology include reproducible production of droplets with adjustable sizing and high placement accuracy, which enable production of individualized dosing even for low dose and high potency drugs. Flexible use of different formulations, such as lipid-based formulations, allows enhancement of the solubility of poorly water soluble and highly lipophilic drugs with DAMPP. Here, DAMPP is used to produce solid oral dosage forms from melts of an active pharmaceutical ingredient and a surfactant. The dosage forms are analyzed to show the amorphous nature, self-emulsifying drug delivery system characteristics and dissolution behavior of these formulations. Copyright © 2017 Elsevier B.V. All rights reserved.

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

PubMed

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

PubMed Central

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

2017-01-01

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. PMID:28981563

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

PubMed

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.

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

PubMed Central

2012-01-01

Background 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. Results 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. Conclusions Multiple lines of evidence from this study suggest that MdANT1 and MdANT2 regulate cell production during fruit growth in

Organization of the Escherichia coli K-12 gene cluster responsible for production of the extracellular polysaccharide colanic acid.

PubMed Central

Stevenson, G; Andrianopoulos, K; Hobbs, M; Reeves, P R

1996-01-01

Colanic acid (CA) is an extracellular polysaccharide produced by most Escherichia coli strains as well as by other species of the family Enterobacteriaceae. We have determined the sequence of a 23-kb segment of the E. coli K-12 chromosome which includes the cluster of genes necessary for production of CA. The CA cluster comprises 19 genes. Two other sequenced genes (orf1.3 and galF), which are situated between the CA cluster and the O-antigen cluster, were shown to be unnecessary for CA production. The CA cluster includes genes for synthesis of GDP-L-fucose, one of the precursors of CA, and the gene for one of the enzymes in this pathway (GDP-D-mannose 4,6-dehydratase) was identified by biochemical assay. Six of the inferred proteins show sequence similarity to glycosyl transferases, and two others have sequence similarity to acetyl transferases. Another gene (wzx) is predicted to encode a protein with multiple transmembrane segments and may function in export of the CA repeat unit from the cytoplasm into the periplasm in a process analogous to O-unit export. The first three genes of the cluster are predicted to encode an outer membrane lipoprotein, a phosphatase, and an inner membrane protein with an ATP-binding domain. Since homologs of these genes are found in other extracellular polysaccharide gene clusters, they may have a common function, such as export of polysaccharide from the cell. PMID:8759852

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

PubMed

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.

Association of polymorphisms of exon 2 of the growth hormone gene with production performance in Huoyan goose.

PubMed

Zhang, Yang; Zhu, Zhen; Xu, Qi; Chen, Guohong

2014-01-07

Primers based on the cDNA sequence of the goose growth hormone (GH) gene in GenBank were designed to amplify exon 2 of the GH gene in Huoyan goose. A total of 552 individuals were brooded in one batch and raised in Liaoning and Jiangsu Provinces, China. Single nucleotide polymorphisms (SNPs) of exon 2 in the GH gene were detected by the polymerase chain reaction (single strand conformation polymorphism method). Homozygotes were subsequently cloned, sequenced and analyzed. Two SNP mutations were detected, and 10 genotypes (referred to as AA, BB, CC, DD, AB, AC, AD, BC, BD and CD) were obtained. Allele D was predominant, and the frequencies of the 10 genotypes fit the Hardy-Weinberg equilibrium in the male, female and whole populations according to the chi-square test. Based on SNP types, the 10 genotypes were combined into three main genotypes. Multiple comparisons were carried out between different genotypes and production traits when the geese were 10 weeks old. Some indices of production performance were significantly (p < 0.05) associated with the genotype. Particularly, geese with genotype AB or BB were highly productive. Thus, these genotypes may serve as selection markers for production traits in Huoyan geese.

Acetate ester production by Chinese yellow rice wine yeast overexpressing the alcohol acetyltransferase-encoding gene ATF2.

PubMed

Zhang, J; Zhang, C; Qi, Y; Dai, L; Ma, H; Guo, X; Xiao, D

2014-11-27

Acetate ester, which are produced by fermenting yeast cells in an enzyme-catalyzed intracellular reaction, are responsible for the fruity character of fermented alcoholic beverages such as Chinese yellow rice wine. Alcohol acetyltransferase (AATase) is currently believed to be the key enzyme responsible for the production of acetate ester. In order to determine the precise role of the ATF2 gene in acetate ester production, an ATF2 gene encoding a type of AATase was overexpressed and the ability of the mutant to form acetate esters (including ethyl acetate, isoamyl acetate, and isobutyl acetate) was investigated. The results showed that after 5 days of fermentation, the concentrations of ethyl acetate, isoamyl acetate, and isobutyl acetate in yellow rice wines fermented with EY2 (pUC-PIA2K) increased to 137.79 mg/L (an approximate 4.9-fold increase relative to the parent cell RY1), 26.68 mg/L, and 7.60 mg/L, respectively. This study confirms that the ATF2 gene plays an important role in the production of acetate ester production during Chinese yellow rice wine fermentation, thereby offering prospects for the development of yellow rice wine yeast starter strains with optimized ester-producing capabilities.

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

PubMed

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.