Prokaryotic expression of chicken interferon-γ fusion protein and its effect on expression of poultry heat shock protein 70 under heat stress.

Science.gov (United States)

Sun, Jinhua; Chen, Yinglin; Qin, Feiyue; Guan, Xueting; Xu, Wei; Xu, Liangmei

2017-06-01

Interferons have attracted considerable attention due to their vital roles in the host immune response and low induction of antibiotic resistance. In this study, total RNA was extracted from spleen cells of chicken embryos inoculated with Newcastle disease vaccine, and the full-length chicken interferon-γ (ChIFN-γ) gene was amplified by RT-PCR. The full complementary DNA sequence of the ChIFN-γ gene was 495 bp long and was cloned into the prokaryotic expression vector pProEX™HT b . The plasmid was transformed into Escherichia coli DH5α and the expression of ChIFN-γ was induced by isopropyl β-D-1-thiogalactopyranoside. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis and Western blot results showed the expressed fusion protein had a molecular weight of approximately 18 kDa and was recognized by an anti-His mAb. Moreover, ChIFN-γ was found to demonstrate anti-viral activity in vitro. To test the in vivo function of ChIFN-γ in broilers under heat stress, a total of 100 broilers were randomly assigned to either a control group or a treated group, in which they were hypodermically injected with recombinant ChIFN-γ. Results demonstrated ChIFN-γ affects the messenger RNA expression levels of heat shock protein 70 (HSP70) in the heart and lung tissues, and decreases the concentration of HSP70 in serum. Therefore, we conclude recombinant ChIFN-γ can reduce heat stress to some extent in vivo. © 2016 Japanese Society of Animal Science.

[Prokaryotic expression of vp3 gene of Muscovy duck parvovirus, and its antiserum preparation for detection of virus multiplication].

Science.gov (United States)

Huang, Yu; Zhu, Yumin; Dong, Shijuan; Yu, Ruisong; Zhang, Yuanshu; Li, Zhen

2015-01-01

New epidemic broke out in recent year which was suspected to be caused by variant Muscovy duck parvovirus (MDPV). For this reason, new MDPV detection methods are needed for the new virus strains. In this study, a pair of primers were designed according to the full-length genome of MDPV strain SAAS-SHNH, which were identified in 2012, and were used to amplify the vp3 gene of MDPV by polymerase chain reaction. After being sequenced, the vp3 gene was subcloned into the prokaryotic expression vector PET28a. The recombinant plasmid was transformed into E. coli BL21 and induced with IPTG. SDS-PAGE and Western blotting analysis showed the MDPV vp3 gene was successfully expressed. After being purified by Ni2+ affinity chromatography system, the recombinant protein was used as antigen to immunize rabbits to obtain antiserum. Western blotting analysis showed that the acquired antiserum could react specifically with VP3 protein of J3D6 strain and MDPV vaccine strain. The antiserum could also be used for detection of cultured MDPV from primary duck embryo fibroblasts by immune fluorescence assay (IFA). It could be concluded that the VP3 protein and its antibody prepared in the research could be used for detection of VP3 antiserum and antigen respectively.

Molecular cloning and expression analysis of a zeta-class ...

African Journals Online (AJOL)

Jane

2011-07-27

Jul 27, 2011 ... sugar-signalling pathway (Chi et al., 2010). All the earlier mentioned ... real-time qPCR analysis was the ABI PRISM7500 real-time PCR system. ... Construction of prokaryotic expression vector of Sc-GST gene. pET29a (+) ...

Cloning, expression, and purification of recombinant protein MPT-64 from a virulent strain of Mycobacterium bovis in a prokaryotic system

Directory of Open Access Journals (Sweden)

Maryam Mohammadi Tashakkori

2016-01-01

Conclusion: These results indicated successful expression and purification of recombinant MPT-64 protein in a prokaryotic system. This protein can be used for serological diagnosis, improved detection of pathogenicity and non-pathogenicity between infected cattle, and for verification of suspected cases of bovine TB.

[Construction and application of prokaryotic expression system of Leptospira interrogans lipL32/1-lipL41/1 fusion gene].

Science.gov (United States)

Luo, Dong-jiao; Yan, Jie; Mao, Ya-fei; Li, Shu-ping; Luo, Yi-hui; Li, Li-wei

2005-01-01

To construct lipL32/1-lipL41/1 fusion gene and its prokaryotic expression system and to determine frequencies of carrying and expression of lipL32 and lipL41 genes in L.interrogans wild strains and specific antibody levels in sera from leptospirosis patients. lipL32/1-lipL41/1 fusion gene was constructed using linking primer PCR method and the prokaryotic expression system of the fusion gene done with routine techniques. SDS-PAGE was used to examine expression of the target recombinant protein rLipL32/1-rLipL41/1. Immunogenicity of rLipL32/1-rLipL41/1 was identified by Western blot. PCR and MAT were performed to detect carrying and expression of lipL32 and lipL41 genes in 97 wild L.interrogans strains. Antibodies against products of lipL32 and lipL41 genes in serum samples from 228 leptospirosis patients were detected by ELISA method. The homogeneity of nucleotide and putative amino acid sequence of lipL32/1-lipL41/1 fusion gene were 99.9 % and 99.8 % in comparison with the reported sequences. Expression output of the target recombinant protein rLipL32/1-rLipL41/1, mainly present in inclusion body, accounted for 10 % of the total bacterial proteins. Both the rabbit antisera against rLipL32/1 and rLipL41/1 could combine to rLipL32/1-rLipL41/1. 97.9 % and 87.6 % of the L.interrogans wild strains had lipL32 and lipL41 genes, respectively. 95.9 % and 84.5 % of the wild strains were positive for MAT with titers of 1:4 - 1:128 using rabbit anti-rLipL32s or anti-rLipL41s sera, respectively. 94.7 % - 97.4 % of the patients'serum samples were positive for rLipL32s antibodies, while 78.5 % - 84.6 % of them were rLipL41s antibodies detectable. lipL32/1-jlipL41/1 fusion gene and its prokaryotic expression system were successfully constructed. The expressed fusion protein had qualified immunogenicity. Both the lipL32 and lipL41 genes are extensively carried and frequently expressed by different serogroups of L.interrogans, and their expression products exhibit cross-antigenicity.

Vectors expressing chimeric Japanese encephalitis dengue 2 viruses.

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Wei, Y; Wang, S; Wang, X

2014-01-01

Vectors based on self-replicating RNAs (replicons) of flaviviruses are becoming powerful tool for expression of heterologous genes in mammalian cells and development of novel antiviral and anticancer vaccines. We constructed two vectors expressing chimeric viruses consisting of attenuated SA14-14-2 strain of Japanese encephalitis virus (JEV) in which the PrM/M-E genes were replaced fully or partially with those of dengue 2 virus (DENV-2). These vectors, named pJED2 and pJED2-1770 were transfected to BHK-21 cells and produced chimeric viruses JED2V and JED2-1770V, respectively. The chimeric viruses could be passaged in C6/36 but not BHK-21 cells. The chimeric viruses produced in C6/36 cells CPE 4-5 days after infection and RT-PCR, sequencing, immunofluorescence assay (IFA) and Western blot analysis confirmed the chimeric nature of produced viruses. The immunogenicity of chimeric viruses in mice was proved by detecting DENV-2 E protein-specific serum IgG antibodies with neutralization titer of 10. Successful preparation of infectious clones of chimeric JEV-DENV-2 viruses showed that JEV-based expression vectors are fully functional.

Geminivirus vectors for high-level expression of foreign proteins in plant cells.

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Mor, Tsafrir S; Moon, Yong-Sun; Palmer, Kenneth E; Mason, Hugh S

2003-02-20

Bean yellow dwarf virus (BeYDV) is a monopartite geminivirus that can infect dicotyledonous plants. We have developed a high-level expression system that utilizes elements of the replication machinery of this single-stranded DNA virus. The replication initiator protein (Rep) mediates release and replication of a replicon from a DNA construct ("LSL vector") that contains an expression cassette for a gene of interest flanked by cis-acting elements of the virus. We used tobacco NT1 cells and biolistic delivery of plasmid DNA for evaluation of replication and expression of reporter genes contained within an LSL vector. By codelivery of a GUS reporter-LSL vector and a Rep-supplying vector, we obtained up to 40-fold increase in expression levels compared to delivery of the reporter-LSL vectors alone. High-copy replication of the LSL vector was correlated with enhanced expression of GUS. Rep expression using a whole BeYDV clone, a cauliflower mosaic virus 35S promoter driving either genomic rep or an intron-deleted rep gene, or 35S-rep contained in the LSL vector all achieved efficient replication and enhancement of GUS expression. We anticipate that this system can be adapted for use in transgenic plants or plant cell cultures with appropriately regulated expression of Rep, with the potential to greatly increase yield of recombinant proteins. Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 430-437, 2003.

Construction of lentiviral shRNA expression vector targeting ...

African Journals Online (AJOL)

DNA oligo was cloned into lentiviral expression vector, and then polymerase chain reaction (PCR) and sequencing analyses were conducted to verify the constructs. The verified vectors were co-transfected into 293FT cells that could produce lentiviral. shRNA lentiviruses from the selected constructs were propagated and ...

Rapid construction of a Bacterial Artificial Chromosomal (BAC) expression vector using designer DNA fragments.

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Chen, Chao; Zhao, Xinqing; Jin, Yingyu; Zhao, Zongbao Kent; Suh, Joo-Won

2014-11-01

Bacterial artificial chromosomal (BAC) vectors are increasingly being used in cloning large DNA fragments containing complex biosynthetic pathways to facilitate heterologous production of microbial metabolites for drug development. To express inserted genes using Streptomyces species as the production hosts, an integration expression cassette is required to be inserted into the BAC vector, which includes genetic elements encoding a phage-specific attachment site, an integrase, an origin of transfer, a selection marker and a promoter. Due to the large sizes of DNA inserted into the BAC vectors, it is normally inefficient and time-consuming to assemble these fragments by routine PCR amplifications and restriction-ligations. Here we present a rapid method to insert fragments to construct BAC-based expression vectors. A DNA fragment of about 130 bp was designed, which contains upstream and downstream homologous sequences of both BAC vector and pIB139 plasmid carrying the whole integration expression cassette. In-Fusion cloning was performed using the designer DNA fragment to modify pIB139, followed by λ-RED-mediated recombination to obtain the BAC-based expression vector. We demonstrated the effectiveness of this method by rapid construction of a BAC-based expression vector with an insert of about 120 kb that contains the entire gene cluster for biosynthesis of immunosuppressant FK506. The empty BAC-based expression vector constructed in this study can be conveniently used for construction of BAC libraries using either microbial pure culture or environmental DNA, and the selected BAC clones can be directly used for heterologous expression. Alternatively, if a BAC library has already been constructed using a commercial BAC vector, the selected BAC vectors can be manipulated using the method described here to get the BAC-based expression vectors with desired gene clusters for heterologous expression. The rapid construction of a BAC-based expression vector facilitates

AAVPG: A vigilant vector where transgene expression is induced by p53

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Bajgelman, Marcio C.; Medrano, Ruan F.V.; Carvalho, Anna Carolina P.V.; Strauss, Bryan E., E-mail: bstrauss@usp.br

2013-12-15

Using p53 to drive transgene expression from viral vectors may provide on demand expression in response to physiologic stress, such as hypoxia or DNA damage. Here we introduce AAVPG, an adeno-associated viral (AAV) vector where a p53-responsive promoter, termed PG, is used to control transgene expression. In vitro assays show that expression from the AAVPG-luc vector was induced specifically in the presence of functional p53 (1038±202 fold increase, p<0.001). The AAVPG-luc vector was an effective biosensor of p53 activation in response to hypoxia (4.48±0.6 fold increase in the presence of 250 µM CoCl{sub 2}, p<0.001) and biomechanical stress (2.53±0.4 fold increase with stretching, p<0.05). In vivo, the vigilant nature of the AAVPG-luc vector was revealed after treatment of tumor-bearing mice with doxorubicin (pre-treatment, 3.4×10{sup 5}±0.43×10{sup 5} photons/s; post-treatment, 6.6×10{sup 5}±2.1×10{sup 5} photons/s, p<0.05). These results indicate that the AAVPG vector is an interesting option for detecting p53 activity both in vitro and in vivo. - Highlights: • AAV vector where transgene expression is controlled by the tumor suppressor p53. • The new vector, AAVPG, shown to function as a biosensor of p53 activity, in vitro and in vivo. • The p53 activity monitored by the AAVPG vector is relevant to cancer and other diseases. • AAVPG reporter gene expression was activated upon DNA damage, hypoxia and mechanical stress.

[Effects of antimicrobial peptide LL-37 expressed and purified from prokaryotes in the murine model of vaginal candidiasis].

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Wang, F; Huo, Y; Yin, L R; Sun, B; Zhang, P P

2016-07-25

To study the effects of antimicrobial peptide LL-37 expressed and purified from prokaryotes on candida albicans growth. (1)Thirty female Kunming mice were treated with estrogen and white candida yeast suspension were poured into vagina to establish a vulvovaginal candidiasis(VVC)murine model. After successful establishing the VVC mouse model, mice were randomly sorted into test group(n=15)and control group(n=15). Suspension(30 μl, 100 μg/ml)of recombinant peptide LL-37 expressed and purified in Prokaryotes was given by intravaginal administration to the test group for 5 days, while the same amount of phosphate buffered saline(PBS)was given to the control group.(2)Tweenty-four hours after treatment, the fungal burden and colony-forming unit(CFU)of vaginal fluids were evaluated. All mice were subsequently sacrificed and vaginal tissues were harvested for tissue homogenate preparation. ELISA was used to determine the levels of nterleukin-10(IL-10)and interferon-γ(IFN-γ)in the isolated vaginal tissues. (1)VVC mouse model was established successfully in this study. Vaginal mucosa congestion, edema, vaginal plica disappearing were obviously observed in the control group. After treatment with recombinant protein LL-37 vaginal mucosa has no obvious change in the test group.(2)Fungal burden and CFU of vaginal fluids were significantly lower in the test group [(4.8±1.0)×10(4) CFU/ml]than that in the control group [(8.5±2.1)×10(4) CFU/ml, P=0.017]. IFN-γ level of the test group was increased [(257±11)vs(197±4)pg/ml, P=0.000], while the level of IL-10 was reduced [(287 ± 15)vs(379 ± 17)pg/ml P=0.000] resulting in a the ratio of IFN-γ/IL-10 was in significantly higher in test group(0.892±0.008 vs 0.496±0.013, P=0.000). Recombinant protein LL-37 expressed and purified from prokaryotes inhibits the growth candida albicans and improves vaginal immunity by adjusting IFN-γ and IL-10 secretion in the VVC mouse model, highlighting the therapeutic potential of LL-37

Construction of PVX virus-expression vector to express enterotoxin ...

African Journals Online (AJOL)

Potato X potyvirus (PVX)-based vector has been comprehensively applied in transient expression system. In order to produce the heterologous proteins more quickly and stably, the ClaI and NotI enzyme sites were introduced into the Enterotoxin fusion gene LTB-ST by polymerase chain reaction (PCR) and the LTB-ST ...

Halophilic & halotolerant prokaryotes in humans.

Science.gov (United States)

Seck, El Hadji; Dufour, Jean-Charles; Raoult, Didier; Lagier, Jean-Christophe

2018-05-04

Halophilic prokaryotes are described as microorganisms living in hypersaline environments. Here, we list the halotolerant and halophilic bacteria which have been isolated in humans. Of the 52 halophilic prokaryotes, 32 (61.54%) were moderately halophilic, 17 (32.69%) were slightly halophilic and three (5.76%) were extremely halophilic prokaryotes. At the phylum level, 29 (54.72%) belong to Firmicutes, 15 (28.84%) to Proteobacteria, four (7.69%) to Actinobacteria, three (5.78%) to Euryarchaeota and one (1.92%) belongs to Bacteroidetes. Halophilic prokaryotes are rarely pathogenic: of these 52 halophilic prokaryotes only two (3.92%) species were classified in Risk Group 2 (Vibrio cholerae, Vibrio parahaemolyticus) and one (1.96%), species in Risk Group 3 (Bacillus anthracis).

Modulating ectopic gene expression levels by using retroviral vectors equipped with synthetic promoters.

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Ferreira, Joshua P; Peacock, Ryan W S; Lawhorn, Ingrid E B; Wang, Clifford L

2011-12-01

The human cytomegalovirus and elongation factor 1α promoters are constitutive promoters commonly employed by mammalian expression vectors. These promoters generally produce high levels of expression in many types of cells and tissues. To generate a library of synthetic promoters capable of generating a range of low, intermediate, and high expression levels, the TATA and CAAT box elements of these promoters were mutated. Other promoter variants were also generated by random mutagenesis. Evaluation using plasmid vectors integrated at a single site in the genome revealed that these various synthetic promoters were capable of expression levels spanning a 40-fold range. Retroviral vectors were equipped with the synthetic promoters and evaluated for their ability to reproduce the graded expression demonstrated by plasmid integration. A vector with a self-inactivating long terminal repeat could neither reproduce the full range of expression levels nor produce stable expression. Using a second vector design, the different synthetic promoters enabled stable expression over a broad range of expression levels in different cell lines. The online version of this article (doi:10.1007/s11693-011-9089-0) contains supplementary material, which is available to authorized users.

Transient foreign gene expression in chloroplasts of cultured tobacco cells after biolistic delivery of chloroplast vectors.

Science.gov (United States)

Daniell, H; Vivekananda, J; Nielsen, B L; Ye, G N; Tewari, K K; Sanford, J C

1990-01-01

Expression of chloramphenicol acetyltransferase (cat) by suitable vectors in chloroplasts of cultured tobacco cells, delivered by high-velocity microprojectiles, is reported here. Several chloroplast expression vectors containing bacterial cat genes, placed under the control of either psbA promoter region from pea (pHD series) or rbcL promoter region from maize (pAC series) have been used in this study. In addition, chloroplast expression vectors containing replicon fragments from pea, tobacco, or maize chloroplast DNA have also been tested for efficiency and duration of cat expression in chloroplasts of tobacco cells. Cultured NT1 tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 (negative control), 35S-CAT (nuclear expression vector), pHD312 (repliconless chloroplast expression vector), and pHD407, pACp18, and pACp19 (chloroplast expression vectors with replicon). Sonic extracts of cells bombarded with pUC118 showed no detectable cat activity in the autoradiograms. Nuclear expression of cat reached two-thirds of the maximal 48 hr after bombardment and the maximal at 72 hr. Cells bombarded with chloroplast expression vectors showed a low level of expression until 48 hr of incubation. A dramatic increase in the expression of cat was observed 24 hr after the addition of fresh medium to cultured cells in samples bombarded with pHD407; the repliconless vector pHD312 showed about 50% of this maximal activity. The expression of nuclear cat and the repliconless chloroplast vector decreased after 72 hr, but a high level of chloroplast cat expression was maintained in cells bombarded with pHD407. Organelle-specific expression of cat in appropriate compartments was checked by introducing various plasmid constructions into tobacco protoplasts by electroporation. Although the nuclear expression vector 35S-CAT showed expression of cat, no activity was observed with any chloroplast vectors.

Three gene expression vector sets for concurrently expressing multiple genes in Saccharomyces cerevisiae.

Science.gov (United States)

Ishii, Jun; Kondo, Takashi; Makino, Harumi; Ogura, Akira; Matsuda, Fumio; Kondo, Akihiko

2014-05-01

Yeast has the potential to be used in bulk-scale fermentative production of fuels and chemicals due to its tolerance for low pH and robustness for autolysis. However, expression of multiple external genes in one host yeast strain is considerably labor-intensive due to the lack of polycistronic transcription. To promote the metabolic engineering of yeast, we generated systematic and convenient genetic engineering tools to express multiple genes in Saccharomyces cerevisiae. We constructed a series of multi-copy and integration vector sets for concurrently expressing two or three genes in S. cerevisiae by embedding three classical promoters. The comparative expression capabilities of the constructed vectors were monitored with green fluorescent protein, and the concurrent expression of genes was monitored with three different fluorescent proteins. Our multiple gene expression tool will be helpful to the advanced construction of genetically engineered yeast strains in a variety of research fields other than metabolic engineering. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Reporter gene expression in fish following cutaneous infection with pantropic retroviral vectors.

Science.gov (United States)

Paul, T A; Burns, J C; Shike, H; Getchell, R; Bowser, P R; Whitlock, K E; Casey, J W

2001-06-01

A central issue in gene delivery systems is choosing promoters that will direct defined and sustainable levels of gene expression. Pantropic retroviral vectors provide a means to insert genes into either somatic or germline cells. In this study, we focused on somatic cell infection by evaluating the activity of 3 promoters inserted by vectors into fish cell lines and fish skin using pantropic retroviruses. In bluegill and zebrafish cell lines, the highest levels of luciferase expression were observed from the 5' murine leukemia virus long terminal repeat of the retroviral vector. The Rous sarcoma virus long terminal repeat and cytomegalovirus early promoter, as internal promoters, generated lower levels of luciferase. Luciferase reporter vectors infected zebrafish skin, as measured by the presence of viral DNA, and expressed luciferase. We infected developing walleye dermal sarcomas with retroviral vectors to provide an environment with enhanced cell proliferation, a condition necessary for integration of the provirus into the host genome. We demonstrated a 4-fold to 7-fold increase in luciferase gene expression in tumor tissue over infections in normal walleye skin.

Three new shuttle vectors for heterologous expression in Zymomonas mobilis

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Qinghua Cao

2016-01-01

Conclusions: These results indicated that these expression vectors are useful tools for gene expression in Z. mobilis and this could provide a solid foundation for further studies of heterologous gene expression in Z. mobilis.

Construction and expression of eukaryotic expression vectors of full-length, amino-terminus and carboxyl-terminus Raf gene

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Zhuomin WANG

2008-06-01

Full Text Available Background and objective Raf is a key molecule in the Ras-Raf-MEK-ERK signal transduction pathway and is highly activated in different human carcinomas. However, its biological functions and regulation mechanisms are still unclear. The aims of this study were to construct eukaryotic expression vectors with Raf full encoding region, truncated amino-terminus and carboxyl-terminus, respectively. Methods Eukaryotic expression vectors of pCMV-Tag2b-Raf-1, pCMV-Tag2b-N-Raf and pCMV-Tag2b-C-Raf were constructed by gene recombination technique and confirmed by restriction enzyme analysis and DNA sequencing. Furthermore, the expression of these fusion proteins was detected by western blot in transient transfected 293T cells. Results The sequences and open reading frames of these three vectors were completely consistent with experimental design. All target proteins can be detected in 293T cells. Conclusion Eukaryotic expression vectors of pCMV-Tag2b-Raf-1, pCMV-Tag2b-N-Raf and pCMV-Tag2b-C-Raf were successfully constructed and can be expressed in 293T cells.

Rule-Based Design of Plant Expression Vectors Using GenoCAD.

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Coll, Anna; Wilson, Mandy L; Gruden, Kristina; Peccoud, Jean

2015-01-01

Plant synthetic biology requires software tools to assist on the design of complex multi-genic expression plasmids. Here a vector design strategy to express genes in plants is formalized and implemented as a grammar in GenoCAD, a Computer-Aided Design software for synthetic biology. It includes a library of plant biological parts organized in structural categories and a set of rules describing how to assemble these parts into large constructs. Rules developed here are organized and divided into three main subsections according to the aim of the final construct: protein localization studies, promoter analysis and protein-protein interaction experiments. The GenoCAD plant grammar guides the user through the design while allowing users to customize vectors according to their needs. Therefore the plant grammar implemented in GenoCAD will help plant biologists take advantage of methods from synthetic biology to design expression vectors supporting their research projects.

Evolution of small prokaryotic genomes

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David José Martínez-Cano

2015-01-01

Full Text Available As revealed by genome sequencing, the biology of prokaryotes with reduced genomes is strikingly diverse. These include free-living prokaryotes with ~800 genes as well as endosymbiotic bacteria with as few as ~140 genes. Comparative genomics is revealing the evolutionary mechanisms that led to these small genomes. In the case of free-living prokaryotes, natural selection directly favored genome reduction, while in the case of endosymbiotic prokaryotes neutral processes played a more prominent role. However, new experimental data suggest that selective processes may be at operation as well for endosymbiotic prokaryotes at least during the first stages of genome reduction. Endosymbiotic prokaryotes have evolved diverse strategies for living with reduced gene sets inside a host-defined medium. These include utilization of host-encoded functions (some of them coded by genes acquired by gene transfer from the endosymbiont and/or other bacteria; metabolic complementation between co-symbionts; and forming consortiums with other bacteria within the host. Recent genome sequencing projects of intracellular mutualistic bacteria showed that previously believed universal evolutionary trends like reduced G+C content and conservation of genome synteny are not always present in highly reduced genomes. Finally, the simplified molecular machinery of some of these organisms with small genomes may be used to aid in the design of artificial minimal cells. Here we review recent genomic discoveries of the biology of prokaryotes endowed with small gene sets and discuss the evolutionary mechanisms that have been proposed to explain their peculiar nature.

Advanced Design of Dumbbell-shaped Genetic Minimal Vectors Improves Non-coding and Coding RNA Expression.

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Jiang, Xiaoou; Yu, Han; Teo, Cui Rong; Tan, Genim Siu Xian; Goh, Sok Chin; Patel, Parasvi; Chua, Yiqiang Kevin; Hameed, Nasirah Banu Sahul; Bertoletti, Antonio; Patzel, Volker

2016-09-01

Dumbbell-shaped DNA minimal vectors lacking nontherapeutic genes and bacterial sequences are considered a stable, safe alternative to viral, nonviral, and naked plasmid-based gene-transfer systems. We investigated novel molecular features of dumbbell vectors aiming to reduce vector size and to improve the expression of noncoding or coding RNA. We minimized small hairpin RNA (shRNA) or microRNA (miRNA) expressing dumbbell vectors in size down to 130 bp generating the smallest genetic expression vectors reported. This was achieved by using a minimal H1 promoter with integrated transcriptional terminator transcribing the RNA hairpin structure around the dumbbell loop. Such vectors were generated with high conversion yields using a novel protocol. Minimized shRNA-expressing dumbbells showed accelerated kinetics of delivery and transcription leading to enhanced gene silencing in human tissue culture cells. In primary human T cells, minimized miRNA-expressing dumbbells revealed higher stability and triggered stronger target gene suppression as compared with plasmids and miRNA mimics. Dumbbell-driven gene expression was enhanced up to 56- or 160-fold by implementation of an intron and the SV40 enhancer compared with control dumbbells or plasmids. Advanced dumbbell vectors may represent one option to close the gap between durable expression that is achievable with integrating viral vectors and short-term effects triggered by naked RNA.

Baculovirus expression vector system: An efficient tool for the ...

African Journals Online (AJOL)

Baculovirus expression vector system is considered one of the most successful and widely acceptable means for the production of recombinant proteins in extremely large quantities. Proper posttranslational modifications of the expressed proteins in insect cells, the usual host of baculoviruses, get them soluble, correctly ...

Transcriptome dynamics-based operon prediction in prokaryotes.

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Fortino, Vittorio; Smolander, Olli-Pekka; Auvinen, Petri; Tagliaferri, Roberto; Greco, Dario

2014-05-16

Inferring operon maps is crucial to understanding the regulatory networks of prokaryotic genomes. Recently, RNA-seq based transcriptome studies revealed that in many bacterial species the operon structure vary with the change of environmental conditions. Therefore, new computational solutions that use both static and dynamic data are necessary to create condition specific operon predictions. In this work, we propose a novel classification method that integrates RNA-seq based transcriptome profiles with genomic sequence features to accurately identify the operons that are expressed under a measured condition. The classifiers are trained on a small set of confirmed operons and then used to classify the remaining gene pairs of the organism studied. Finally, by linking consecutive gene pairs classified as operons, our computational approach produces condition-dependent operon maps. We evaluated our approach on various RNA-seq expression profiles of the bacteria Haemophilus somni, Porphyromonas gingivalis, Escherichia coli and Salmonella enterica. Our results demonstrate that, using features depending on both transcriptome dynamics and genome sequence characteristics, we can identify operon pairs with high accuracy. Moreover, the combination of DNA sequence and expression data results in more accurate predictions than each one alone. We present a computational strategy for the comprehensive analysis of condition-dependent operon maps in prokaryotes. Our method can be used to generate condition specific operon maps of many bacterial organisms for which high-resolution transcriptome data is available.

Sleeping Beauty-baculovirus hybrid vectors for long-term gene expression in the eye.

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Turunen, Tytteli Anni Kaarina; Laakkonen, Johanna Päivikki; Alasaarela, Laura; Airenne, Kari Juhani; Ylä-Herttuala, Seppo

2014-01-01

A baculovirus vector is capable of efficiently transducing many nondiving and diving cell types. However, the potential of baculovirus is restricted for many gene delivery applications as a result of the transient gene expression that it mediates. The plasmid-based Sleeping Beauty (SB) transposon system integrates transgenes into target cell genome efficiently with a genomic integration pattern that is generally considered safer than the integration of many other integrating vectors; yet efficient delivery of therapeutic genes into cells of target tissues in vivo is a major challenge for nonviral gene therapy. In the present study, SB was introduced into baculovirus to obtain novel hybrid vectors that would combine the best features of the two vector systems (i.e. effective gene delivery and efficient integration into the genome), thus circumventing the major limitations of these vectors. We constructed and optimized SB-baculovirus hybrid vectors that bear either SB100x transposase or SB transposon in the forward or reverse orientations with respect to the viral backbone The functionality of the novel hybrid vectors was investigated in cell cultures and in a proof-of-concept study in the mouse eye. The hybrid vectors showed high and sustained transgene expression that remained stable and demonstrated no signs of decline during the 2 months follow-up in vitro. These results were verified in the mouse eye where persistent transgene expression was detected two months after intravitreal injection. Our results confirm that (i) SB-baculovirus hybrid vectors mediate long-term gene expression in vitro and in vivo, and (ii) the hybrid vectors are potential new tools for the treatment of ocular diseases. Copyright © 2014 John Wiley & Sons, Ltd.

Construction of an expression vector for Lactococcus lactis based on ...

African Journals Online (AJOL)

To construct an expression vector for Lactococcus lactis, the EmPMT fragment which contained the erythromycin resistance gene, P32 promoter, multiple cloning site (MCS) and terminator (T) was subcloned into the small cryptic plasmid pAR141. The resulting vector, designated as pAR1411, was found to be stably ...

A versatile system for USER cloning-based assembly of expression vectors for mammalian cell engineering.

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Anne Mathilde Lund

Full Text Available A new versatile mammalian vector system for protein production, cell biology analyses, and cell factory engineering was developed. The vector system applies the ligation-free uracil-excision based technique--USER cloning--to rapidly construct mammalian expression vectors of multiple DNA fragments and with maximum flexibility, both for choice of vector backbone and cargo. The vector system includes a set of basic vectors and a toolbox containing a multitude of DNA building blocks including promoters, terminators, selectable marker- and reporter genes, and sequences encoding an internal ribosome entry site, cellular localization signals and epitope- and purification tags. Building blocks in the toolbox can be easily combined as they contain defined and tested Flexible Assembly Sequence Tags, FASTs. USER cloning with FASTs allows rapid swaps of gene, promoter or selection marker in existing plasmids and simple construction of vectors encoding proteins, which are fused to fluorescence-, purification-, localization-, or epitope tags. The mammalian expression vector assembly platform currently allows for the assembly of up to seven fragments in a single cloning step with correct directionality and with a cloning efficiency above 90%. The functionality of basic vectors for FAST assembly was tested and validated by transient expression of fluorescent model proteins in CHO, U-2-OS and HEK293 cell lines. In this test, we included many of the most common vector elements for heterologous gene expression in mammalian cells, in addition the system is fully extendable by other users. The vector system is designed to facilitate high-throughput genome-scale studies of mammalian cells, such as the newly sequenced CHO cell lines, through the ability to rapidly generate high-fidelity assembly of customizable gene expression vectors.

pEPito: a significantly improved non-viral episomal expression vector for mammalian cells

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Ogris Manfred

2010-03-01

Full Text Available Abstract Background The episomal replication of the prototype vector pEPI-1 depends on a transcription unit starting from the constitutively expressed Cytomegalovirus immediate early promoter (CMV-IEP and directed into a 2000 bp long matrix attachment region sequence (MARS derived from the human β-interferon gene. The original pEPI-1 vector contains two mammalian transcription units and a total of 305 CpG islands, which are located predominantly within the vector elements necessary for bacterial propagation and known to be counterproductive for persistent long-term transgene expression. Results Here, we report the development of a novel vector pEPito, which is derived from the pEPI-1 plasmid replicon but has considerably improved efficacy both in vitro and in vivo. The pEPito vector is significantly reduced in size, contains only one transcription unit and 60% less CpG motives in comparison to pEPI-1. It exhibits major advantages compared to the original pEPI-1 plasmid, including higher transgene expression levels and increased colony-forming efficiencies in vitro, as well as more persistent transgene expression profiles in vivo. The performance of pEPito-based vectors was further improved by replacing the CMV-IEP with the human CMV enhancer/human elongation factor 1 alpha promoter (hCMV/EF1P element that is known to be less affected by epigenetic silencing events. Conclusions The novel vector pEPito can be considered suitable as an improved vector for biotechnological applications in vitro and for non-viral gene delivery in vivo.

Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression.

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Trinh, Alice T; Ball, Bret G; Weber, Erin; Gallaher, Timothy K; Gluzman-Poltorak, Zoya; Anderson, French; Basile, Lena A

2009-12-30

Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone. A MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection. Vectors that contained the ADA LCR were preferentially expressed in T-cell lines. Further improvements

Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression

Science.gov (United States)

2009-01-01

Background Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone. Methods A MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection. Results Vectors that contained the ADA LCR were preferentially expressed in T

Optimized expression and purification of NavAb provide the structural insight into the voltage dependence.

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Irie, Katsumasa; Haga, Yukari; Shimomura, Takushi; Fujiyoshi, Yoshinori

2018-01-01

Voltage-gated sodium channels are crucial for electro-signalling in living systems. Analysis of the molecular mechanism requires both fine electrophysiological evaluation and high-resolution channel structures. Here, we optimized a dual expression system of NavAb, which is a well-established standard of prokaryotic voltage-gated sodium channels, for E. coli and insect cells using a single plasmid vector to analyse high-resolution protein structures and measure large ionic currents. Using this expression system, we evaluated the voltage dependence and determined the crystal structures of NavAb wild-type and two mutants, E32Q and N49K, whose voltage dependence were positively shifted and essential interactions were lost in voltage sensor domain. The structural and functional comparison elucidated the molecular mechanisms of the voltage dependence of prokaryotic voltage-gated sodium channels. © 2017 Federation of European Biochemical Societies.

Translational selection is ubiquitous in prokaryotes.

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Fran Supek

2010-06-01

Full Text Available Codon usage bias in prokaryotic genomes is largely a consequence of background substitution patterns in DNA, but highly expressed genes may show a preference towards codons that enable more efficient and/or accurate translation. We introduce a novel approach based on supervised machine learning that detects effects of translational selection on genes, while controlling for local variation in nucleotide substitution patterns represented as sequence composition of intergenic DNA. A cornerstone of our method is a Random Forest classifier that outperformed previous distance measure-based approaches, such as the codon adaptation index, in the task of discerning the (highly expressed ribosomal protein genes by their codon frequencies. Unlike previous reports, we show evidence that translational selection in prokaryotes is practically universal: in 460 of 461 examined microbial genomes, we find that a subset of genes shows a higher codon usage similarity to the ribosomal proteins than would be expected from the local sequence composition. These genes constitute a substantial part of the genome--between 5% and 33%, depending on genome size--while also exhibiting higher experimentally measured mRNA abundances and tending toward codons that match tRNA anticodons by canonical base pairing. Certain gene functional categories are generally enriched with, or depleted of codon-optimized genes, the trends of enrichment/depletion being conserved between Archaea and Bacteria. Prominent exceptions from these trends might indicate genes with alternative physiological roles; we speculate on specific examples related to detoxication of oxygen radicals and ammonia and to possible misannotations of asparaginyl-tRNA synthetases. Since the presence of codon optimizations on genes is a valid proxy for expression levels in fully sequenced genomes, we provide an example of an "adaptome" by highlighting gene functions with expression levels elevated specifically in

A family of E. coli expression vectors for laboratory scale and high throughput soluble protein production

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Bottomley Stephen P

2006-03-01

Full Text Available Abstract Background In the past few years, both automated and manual high-throughput protein expression and purification has become an accessible means to rapidly screen and produce soluble proteins for structural and functional studies. However, many of the commercial vectors encoding different solubility tags require different cloning and purification steps for each vector, considerably slowing down expression screening. We have developed a set of E. coli expression vectors with different solubility tags that allow for parallel cloning from a single PCR product and can be purified using the same protocol. Results The set of E. coli expression vectors, encode for either a hexa-histidine tag or the three most commonly used solubility tags (GST, MBP, NusA and all with an N-terminal hexa-histidine sequence. The result is two-fold: the His-tag facilitates purification by immobilised metal affinity chromatography, whilst the fusion domains act primarily as solubility aids during expression, in addition to providing an optional purification step. We have also incorporated a TEV recognition sequence following the solubility tag domain, which allows for highly specific cleavage (using TEV protease of the fusion protein to yield native protein. These vectors are also designed for ligation-independent cloning and they possess a high-level expressing T7 promoter, which is suitable for auto-induction. To validate our vector system, we have cloned four different genes and also one gene into all four vectors and used small-scale expression and purification techniques. We demonstrate that the vectors are capable of high levels of expression and that efficient screening of new proteins can be readily achieved at the laboratory level. Conclusion The result is a set of four rationally designed vectors, which can be used for streamlined cloning, expression and purification of target proteins in the laboratory and have the potential for being adaptable to a high

Construction of novel shuttle expression vectors for gene expression in Bacillus subtilis and Bacillus pumilus.

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Shao, Huanhuan; Cao, Qinghua; Zhao, Hongyan; Tan, Xuemei; Feng, Hong

2015-01-01

A native plasmid (pSU01) was detected by genome sequencing of Bacillus subtilis strain S1-4. Two pSU01-based shuttle expression vectors pSU02-AP and pSU03-AP were constructed enabling stable replication in B. subtilis WB600. These vectors contained the reporter gene aprE, encoding an alkaline protease from Bacillus pumilus BA06. The expression vector pSU03-AP only possessed the minimal replication elements (rep, SSO, DSO) and exhibited more stability on structure, suggesting that the rest of the genes in pSU01 (ORF1, ORF2, mob, hsp) were unessential for the structural stability of plasmid in B. subtilis. In addition, recombinant production of the alkaline protease was achieved more efficiently with pSU03-AP whose copy number was estimated to be more than 100 per chromosome. Furthermore, pSU03-AP could also be used to transform and replicate in B. pumilus BA06 under selective pressure. In conclusion, pSU03-AP is expected to be a useful tool for gene expression in Bacillus subtilis and B. pumilus.

Glycoprotein is enough for sindbis virus-derived DNA vector to express heterogenous genes

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Fu Juanjuan

2011-07-01

Full Text Available Abstract To investigate the necessity and potential application of structural genes for expressing heterogenous genes from Sindbis virus-derived vector, the DNA-based expression vector pVaXJ was constructed by placing the recombinant genome of sindbis-like virus XJ-160 under the control of the human cytomegalovirus (CMV promoter of the plasmid pVAX1, in which viral structural genes were replaced by a polylinker cassette to allow for insertion of heterologous genes. The defect helper plasmids pVaE or pVaC were developed by cloning the gene of glycoprotein E3E26KE1 or capsid protein of XJ-160 virus into pVAX1, respectively. The report gene cassette pVaXJ-EGFP or pV-Gluc expressing enhanced green fluorescence protein (EGFP or Gaussia luciferase (G.luc were constructed by cloning EGFP or G.luc gene into pVaXJ. EGFP or G.luc was expressed in the BHK-21 cells co-transfected with report gene cassettes and pVaE at levels that were comparable to those produced by report gene cassettes, pVaC and pVaE and were much higher than the levels produced by report gene cassette and pVaC, suggesting that glycoprotein is enough for Sindbis virus-derived DNA vector to express heterogenous genes in host cells. The method of gene expression from Sindbis virus-based DNA vector only co-transfected with envelop E gene increase the conveniency and the utility of alphavirus-based vector systems in general.

Multigenic lentiviral vectors for combined and tissue-specific expression of miRNA- and protein-based antiangiogenic factors

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Anne Louise Askou

Full Text Available Lentivirus-based gene delivery vectors carrying multiple gene cassettes are powerful tools in gene transfer studies and gene therapy, allowing coexpression of multiple therapeutic factors and, if desired, fluorescent reporters. Current strategies to express transgenes and microRNA (miRNA clusters from a single vector have certain limitations that affect transgene expression levels and/or vector titers. In this study, we describe a novel vector design that facilitates combined expression of therapeutic RNA- and protein-based antiangiogenic factors as well as a fluorescent reporter from back-to-back RNApolII-driven expression cassettes. This configuration allows effective production of intron-embedded miRNAs that are released upon transduction of target cells. Exploiting such multigenic lentiviral vectors, we demonstrate robust miRNA-directed downregulation of vascular endothelial growth factor (VEGF expression, leading to reduced angiogenesis, and parallel impairment of angiogenic pathways by codelivering the gene encoding pigment epithelium-derived factor (PEDF. Notably, subretinal injections of lentiviral vectors reveal efficient retinal pigment epithelium-specific gene expression driven by the VMD2 promoter, verifying that multigenic lentiviral vectors can be produced with high titers sufficient for in vivo applications. Altogether, our results suggest the potential applicability of combined miRNA- and protein-encoding lentiviral vectors in antiangiogenic gene therapy, including new combination therapies for amelioration of age-related macular degeneration.

Transient foreign gene expression in chloroplasts of cultured tobacco cells after biolistic delivery of chloroplast vectors.

OpenAIRE

Daniell, H; Vivekananda, J; Nielsen, B L; Ye, G N; Tewari, K K; Sanford, J C

1990-01-01

Expression of chloramphenicol acetyltransferase (cat) by suitable vectors in chloroplasts of cultured tobacco cells, delivered by high-velocity microprojectiles, is reported here. Several chloroplast expression vectors containing bacterial cat genes, placed under the control of either psbA promoter region from pea (pHD series) or rbcL promoter region from maize (pAC series) have been used in this study. In addition, chloroplast expression vectors containing replicon fragments from pea, tobacc...

A framework for classification of prokaryotic protein kinases.

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Nidhi Tyagi

Full Text Available BACKGROUND: Overwhelming majority of the Serine/Threonine protein kinases identified by gleaning archaeal and eubacterial genomes could not be classified into any of the well known Hanks and Hunter subfamilies of protein kinases. This is owing to the development of Hanks and Hunter classification scheme based on eukaryotic protein kinases which are highly divergent from their prokaryotic homologues. A large dataset of prokaryotic Serine/Threonine protein kinases recognized from genomes of prokaryotes have been used to develop a classification framework for prokaryotic Ser/Thr protein kinases. METHODOLOGY/PRINCIPAL FINDINGS: We have used traditional sequence alignment and phylogenetic approaches and clustered the prokaryotic kinases which represent 72 subfamilies with at least 4 members in each. Such a clustering enables classification of prokaryotic Ser/Thr kinases and it can be used as a framework to classify newly identified prokaryotic Ser/Thr kinases. After series of searches in a comprehensive sequence database we recognized that 38 subfamilies of prokaryotic protein kinases are associated to a specific taxonomic level. For example 4, 6 and 3 subfamilies have been identified that are currently specific to phylum proteobacteria, cyanobacteria and actinobacteria respectively. Similarly subfamilies which are specific to an order, sub-order, class, family and genus have also been identified. In addition to these, we also identify organism-diverse subfamilies. Members of these clusters are from organisms of different taxonomic levels, such as archaea, bacteria, eukaryotes and viruses. CONCLUSION/SIGNIFICANCE: Interestingly, occurrence of several taxonomic level specific subfamilies of prokaryotic kinases contrasts with classification of eukaryotic protein kinases in which most of the popular subfamilies of eukaryotic protein kinases occur diversely in several eukaryotes. Many prokaryotic Ser/Thr kinases exhibit a wide variety of modular

[Prokaryotic expression of recombinant prochymosin gene and its antiserum preparation].

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Li, Xin-ping; Liu, Huan-huan; Pu, Yan; Zhang, Fu-chun; Li, Yi-jie

2012-07-01

To optimize the prochymosin (pCHY) gene codons and express the gene in Escherichia coli (E.coli), and to prepare its antiserum and detect chymosin protein specifically. According to codon usage bias of E.coli, prochymosin gene sequence was synthesized based on the conserved sequences of prochymosin gene from bovine, lamb and camel, and then cloned into the plasmid pET-30a and pcDNA3-AAT-COMP-C3d3 (pcD-ACC), respectively. pET-30a-pCHY was expressed, as the detected antigen, in E.coli BL21(DE3) after IPTG induction. RT-PCR was used to detect prochymosin mRNA expression in liver from the mice injected pcDNA3-AAT-COMP-pCHY-C3d3(pACCC) by hydrodynamics-based transfection method. To prepare the antiserum of prochymosin, pACCC and GST-pCHY proteins were used to immunize New Zealand rabbits in accordance with DNA prime-protein boost strategy. Antibody levels were tested by ELISA. Western blotting showed the molecular weight of His-pCHY protein was about 55 000, similar to the expected molecular size. ELISA demonstrated that the titer level of prochymosin antiserum was high. Based on the codon optimization, we have obtained high-titer prochymosin antiserum through DNA vaccine vector pcD-ACC combined with DNA prime-protein boost strategy, similar to that by protein vaccine.

EMMA: An Extensible Mammalian Modular Assembly Toolkit for the Rapid Design and Production of Diverse Expression Vectors.

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Martella, Andrea; Matjusaitis, Mantas; Auxillos, Jamie; Pollard, Steven M; Cai, Yizhi

2017-07-21

Mammalian plasmid expression vectors are critical reagents underpinning many facets of research across biology, biomedical research, and the biotechnology industry. Traditional cloning methods often require laborious manual design and assembly of plasmids using tailored sequential cloning steps. This process can be protracted, complicated, expensive, and error-prone. New tools and strategies that facilitate the efficient design and production of bespoke vectors would help relieve a current bottleneck for researchers. To address this, we have developed an extensible mammalian modular assembly kit (EMMA). This enables rapid and efficient modular assembly of mammalian expression vectors in a one-tube, one-step golden-gate cloning reaction, using a standardized library of compatible genetic parts. The high modularity, flexibility, and extensibility of EMMA provide a simple method for the production of functionally diverse mammalian expression vectors. We demonstrate the value of this toolkit by constructing and validating a range of representative vectors, such as transient and stable expression vectors (transposon based vectors), targeting vectors, inducible systems, polycistronic expression cassettes, fusion proteins, and fluorescent reporters. The method also supports simple assembly combinatorial libraries and hierarchical assembly for production of larger multigenetic cargos. In summary, EMMA is compatible with automated production, and novel genetic parts can be easily incorporated, providing new opportunities for mammalian synthetic biology.

Algevir: An Expression System for Microalgae Based on Viral Vectors

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Bernardo Bañuelos-Hernández

2017-06-01

Full Text Available The use of recombinant algae for the production of valuable compounds is opening promising biotechnological applications. However, the development of efficient expression approaches is still needed to expand the exploitation of microalgae in biotechnology. Herein, the concept of using viral expression vectors in microalgae was explored for the first time. An inducible geminiviral vector leading to Rep-mediated replication of the expression cassette allowed the production of antigenic proteins at high levels. This system, called Algevir, allows the production of complex viral proteins (GP1 from Zaire ebolavirus and bacterial toxin subunits (B subunit of the heat-labile Escherichia coli enterotoxin, which retained their antigenic activity. The highest achieved yield was 1.25 mg/g fresh biomass (6 mg/L of culture, which was attained 3 days after transformation. The Algevir system allows for a fast and efficient production of recombinant proteins, overcoming the difficulties imposed by the low yields and unstable expression patterns frequently observed in stably transformed microalgae at the nuclear level; as well as the toxicity of some target proteins.

Algevir: An Expression System for Microalgae Based on Viral Vectors

Science.gov (United States)

Bañuelos-Hernández, Bernardo; Monreal-Escalante, Elizabeth; González-Ortega, Omar; Angulo, Carlos; Rosales-Mendoza, Sergio

2017-01-01

The use of recombinant algae for the production of valuable compounds is opening promising biotechnological applications. However, the development of efficient expression approaches is still needed to expand the exploitation of microalgae in biotechnology. Herein, the concept of using viral expression vectors in microalgae was explored for the first time. An inducible geminiviral vector leading to Rep-mediated replication of the expression cassette allowed the production of antigenic proteins at high levels. This system, called Algevir, allows the production of complex viral proteins (GP1 from Zaire ebolavirus) and bacterial toxin subunits (B subunit of the heat-labile Escherichia coli enterotoxin), which retained their antigenic activity. The highest achieved yield was 1.25 mg/g fresh biomass (6 mg/L of culture), which was attained 3 days after transformation. The Algevir system allows for a fast and efficient production of recombinant proteins, overcoming the difficulties imposed by the low yields and unstable expression patterns frequently observed in stably transformed microalgae at the nuclear level; as well as the toxicity of some target proteins. PMID:28713333

Prolonged liver-specific transgene expression by a non-primate lentiviral vector

International Nuclear Information System (INIS)

Condiotti, Reba; Curran, Michael A.; Nolan, Garry P.; Giladi, Hilla; Ketzinel-Gilad, Mali; Gross, Eitan; Galun, Eithan

2004-01-01

Liver-directed gene therapy has the potential for treatment of numerous inherited diseases affecting metabolic functions. The aim of this study was to evaluate gene expression in hepatocytes using feline immunodeficiency virus-based lentiviral vectors, which may be potentially safer than those based on human immunodeficiency virus. In vitro studies revealed that gene expression was stable for up to 24 days post-transduction and integration into the host cell genome was suggested by Alu PCR and Southern blot analyses. Systemic in vivo administration of viral particles by the hydrodynamics method resulted in high levels of gene expression exclusively in the liver for over 7 months whereas injection of plasmid DNA by the same method led to transient expression levels. Our studies suggest that feline immunodeficiency-based lentiviral vectors specifically transduce liver cells and may be used as a novel vehicle of gene delivery for treatment of metabolic disease

Transgene Expression and Host Cell Responses to Replication-Defective, Single-Cycle, and Replication-Competent Adenovirus Vectors

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Catherine M. Crosby

2017-02-01

Full Text Available Most adenovirus (Ad vectors are E1 gene deleted replication defective (RD-Ad vectors that deliver one transgene to the cell and all expression is based on that one gene. In contrast, E1-intact replication-competent Ad (RC-Ad vectors replicate their DNA and their transgenes up to 10,000-fold, amplifying transgene expression markedly higher than RD-Ad vectors. While RC-Ad are more potent, they run the real risk of causing adenovirus infections in vector recipients and those that administer them. To gain the benefits of transgene amplification, but avoid the risk of Ad infections, we developed “single cycle” Ad (SC-Ad vectors. SC-Ads amplify transgene expression and generated markedly stronger and more persistent immune responses than RD-Ad as expected. However, they also unexpectedly generated stronger immune responses than RC-Ad vectors. To explore the basis of this potency here, we compared gene expression and the cellular responses to infection to these vectors in vitro and in vivo. In vitro, in primary human lung epithelial cells, SC- and RC-Ad amplified their genomes more than 400-fold relative to RD-Ad with higher replication by SC-Ad. This replication translated into higher green fluorescent protein (GFP expression for 48 h by SC- and RC-Ad than by RD-Ad. In vitro, in the absence of an immune system, RD-Ad expression became higher by 72 h coincident with cell death mediated by SC- and RC-Ad and release of transgene product from the dying cells. When the vectors were compared in human THP-1 Lucia- interferon-stimulated gene (ISG cells, which are a human monocyte cell line that have been modified to quantify ISG activity, RC-Ad6 provoked significantly stronger ISG responses than RD- or SC-Ad. In mice, intravenous or intranasal injection produced up to 100-fold genome replication. Under these in vivo conditions in the presence of the immune system, luciferase expression by RC and SC-Ad was markedly higher than that by RD-Ad. In

Expression of Separate Proteins in the Same Plant Leaves and Cells Using Two Independent Virus-Based Gene Vectors

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Maria R. Mendoza

2017-11-01

Full Text Available Plant viral vectors enable the expression of proteins at high levels in a relatively short time. For many purposes (e.g., cell biological interaction studies it may be desirable to express more than one protein in a single cell but that is often not feasible when using a single virus vector. Such a co-expression strategy requires the simultaneous delivery by two compatible and non-competitive viruses that can co-exist to each express a separate protein. Here, we report on the use of two agro-launchable coat-protein gene substitution GFP-expressing virus vector systems based on Tomato bushy stunt virus (TBSV referred to as TG, and Tobacco mosaic virus (TMV annotated as TRBO-G. TG expressed GFP in Nicotiana benthamiana, tomato, lettuce and cowpea, whereas expression from TRBO-G was detected only in the first two species. Upon co-infiltration of the two vectors co-expression was monitored by: molecular detection of the two slightly differently sized GFPs, suppressor-complementation assays, and using TG in combination with TRBO-RFP. All the results revealed that in N. benthamiana and tomato the TBSV and TMV vectors accumulated and expressed proteins in the same plants, the same leaves, and in the same cells. Therefore, co-expression by these two vectors provides a platform for fast and high level expression of proteins to study their cell biology or other properties.

Development of new USER-based cloning vectors for multiple genes expression in Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Kildegaard, Kanchana Rueksomtawin; Jensen, Niels Bjerg; Maury, Jerome

2013-01-01

auxotrophic and dominant markers for convenience of use. Our vector set also contains both integrating and multicopy vectors for stability of protein expression and high expression level. We will make the new vector system available to the yeast community and provide a comprehensive protocol for cloning...... the production strain with the proper phenotype and product yield. However, the sequential number of metabolic engineering is time-consuming. Furthermore, the number of available selectable markers is also limiting the number of genetic modifications. To overcome these limitations, we have developed a new set...... of shuttle vectors for convenience of use for high-throughput cloning and selectable marker recycling. The new USER-based cloning vectors consist of a unique USER site and a CRE-loxP-mediated marker recycling system. The USER site allows insertion of genes of interest along with a bidirectional promoter...

Construction of permanently inducible miRNA-based expression vectors using site-specific recombinases

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Garwick-Coppens Sara E

2011-11-01

Full Text Available Abstract Background RNA interference (RNAi is a conserved gene silencing mechanism mediated by small inhibitory microRNAs (miRNAs. Promoter-driven miRNA expression vectors have emerged as important tools for delivering natural or artificially designed miRNAs to eukaryotic cells and organisms. Such systems can be used to query the normal or pathogenic functions of natural miRNAs or messenger RNAs, or to therapeutically silence disease genes. Results As with any molecular cloning procedure, building miRNA-based expression constructs requires a time investment and some molecular biology skills. To improve efficiency and accelerate the construction process, we developed a method to rapidly generate miRNA expression vectors using recombinases instead of more traditional cut-and-paste molecular cloning techniques. In addition to streamlining the construction process, our cloning strategy provides vectors with added versatility. In our system, miRNAs can be constitutively expressed from the U6 promoter, or inducibly expressed by Cre recombinase. We also engineered a built-in mechanism to destroy the vector with Flp recombinase, if desired. Finally, to further simplify the construction process, we developed a software package that automates the prediction and design of optimal miRNA sequences using our system. Conclusions We designed and tested a modular system to rapidly clone miRNA expression cassettes. Our strategy reduces the hands-on time required to successfully generate effective constructs, and can be implemented in labs with minimal molecular cloning expertise. This versatile system provides options that permit constitutive or inducible miRNA expression, depending upon the needs of the end user. As such, it has utility for basic or translational applications.

Construction and identification of eukaryotic expression vector of pcDNA3-UHRF1

International Nuclear Information System (INIS)

Li Xinli; Zhu Ran; Zhu Wei; Fan Saijun; Meng Qinghui

2011-01-01

Objective: To generate eukaryotic expression vector of pcDNA3-UHRF1(ubiquitin-like, containing PHD and RING finger domains 1, UHRF1) and testify its expression in breast cancer cells MDA-MB-231. Methods: A 2.3 kb cDNA fragment was amplified from the total RNA of the human breast cancer cells MCF-7 by the RT-PCR method and was cloned into the plasmid pcDNA3. The vector was identified by the double digestion with restriction enzymes Kpn I and Xho I and was sequenced. The cDNA of UHRF1 was transfected into human breast cancer cells MDA-MB-231 by Lipofactamin2000. The positive clones were selected by G418. The expression of the UHRF1 was detected by RT-PCR and Western blot analysis. Results: The recombinant eukaryotic expression vector pcDNA3-UHRF1 was digested with Kpn I and BamH I, and the electrophoresis of the digested products showed two fragments; 2.3kb fragment of UHRF1 and 5.4 kb fragment of pcDNA3, and the sequence inserted was identical to the published sequence. The MDA-MB-231 cells transfected with the pcDNA3-UHRF1 plasmid expressed a high level of the UHRF1 mRNA and protein. Conclusion: The recombinant eukaryotic cell expression vector of pcDNA3-UHRF1 is constructed successfully. The recombinant plasmid pcDNA3-UHRF1 can provide a very useful tool and lay an important foundation for the research on the function of UHRF1. (authors)

Over-expression of gene encoding heat shock protein 70 from Mycobacterium tuberculosis and its evaluation as vaccine adjuvant

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J Dhakal

2013-01-01

Full Text Available Background: Heat shock proteins (Hsps are evolutionary ancient and highly conserved molecular chaperons found in prokaryotes as well as eukaryotes. Hsp70 is a predominant member of Hsp family. Microbial Hsp70s (mHsp70s have acquired special significance in immunity since they have been shown to be potent activators of the innate immune system and generate specific immune responses against tumours and infectious agents. Objectives: The present study was aimed to clone express and purify recombinant Hsp70 from the Mycobacterium tuberculosis and characterise it immunologically. The study also aimed at determining the potential of recombinant M. tuberculosis heat shock protein (rMTB-Hsp70 as adjuvant or antigen carrier. Materials and Methods: Cloning of M. tuberculosis heat shock protein (MTB-Hsp70 amplicon was carried out using the pGEMT-Easy vector although for expression, pProExHTb prokaryotic expression vector was used. Purification of recombinant Hsp70 was carried out by nickel-nitrilotriacetic acid (Ni-NTA affinity chromatography. For immunological characterization and determining the adjuvant effect of MTB-Hsp70, BALB/c mice were used. The data obtained was statistically analysed. Results: Hsp70 gene was cloned, sequenced and the sequence data were submitted to National Center for Biotechnology Information (NCBI. Recombinant MTB-Hsp70 was successfully over-expressed using the prokaryotic expression system and purified to homogeneity. The protein was found to be immunodominant. Significant adjuvant effect was produced by the rMTB-Hsp70 when inoculated with recombinant outer membrane protein 31; however, effect was less than the conventionally used the Freund′s adjuvant. Conclusion: Protocol standardised can be followed for bulk production of rHsp70 in a cost-effective manner. Significant adjuvant effect was produced by rMTB-Hsp70; however, the effect was than Freund′s adjuvant. Further, studies need to be carried out to explore its

Integrating ribosomal promoter vectors that offer a choice of constitutive expression profiles in Leishmania donovani.

Science.gov (United States)

Soysa, Radika; Tran, Khoa D; Ullman, Buddy; Yates, Phillip A

2015-12-01

We have designed a novel series of integrating ribosomal RNA promoter vectors with five incrementally different constitutive expression profiles, covering a 250-fold range. Differential expression was achieved by placing different combinations of synthetic or leishmanial DNA sequences upstream and downstream of the transgene coding sequence in order to modulate pre-mRNA processing efficiency and mRNA stability, respectively. All of the vectors have extensive multiple cloning sites, and versions are available for producing N- or C- terminal GFP fusions at each of the possible relative expression levels. In addition, the modular configuration of the vectors allows drug resistance cassettes and other components to be readily exchanged. In toto, these vectors should be useful additions to the toolkit available for molecular and genetic studies of Leishmania donovani. Copyright © 2016 Elsevier B.V. All rights reserved.

Construction of rat beta defensin-2 eukaryotic expression vector and expression in the transfected rat corneal epithelial cell

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Jing Dan

2017-03-01

Full Text Available AIM: To construct a recombinant eukaryotic expression vector of rat beta defensin-2(rBD-2, transfect it into the rat corneal epithelial cells with lipofection, determine the expression of target gene in the transfected cells, and discuss the potentiality of recombinant plasmid expressed in corneal epithelial cells, hoping to provide an experimental foundation for further study on the antimicrobial activity of rBD-2 in vitro and in vivo and to assess the probability of defensins as a new application for infectious corneal diseases in the future. METHODS: The synthetic rBD-2 DNA fragment was inserted between the XhoI and BamHI restriction enzyme cutting sites of eukaryotic expression vector pIRES2-ZsGreen1 to construct the recombinant plasmid pIRES2-ZsGreen1-rBD-2, then transformed it into E.coli DH5α, positive clones were screened by kanamycin and identified with restriction endonucleases and sequencing analysis. Transfection into the rat corneal epithelial cells was performed by lipofection. Then the experiment was divided into three groups: rat corneal epithelial cell was transfected with the recombinant plasmid pIRES2- ZsGreen1-rBD-2, rat corneal epithelial cell was transfected with the empty plasmid pIRES2-ZsGreen1 and the non-transfected group. The inverted fluorescence microscope was used to observe the transfection process. At last, the level of rBD-2 mRNA expressed in the transfected cells and the control groups are compared by the real-time fluoresence relative quantitative PCR. RESULTS: The recombinant eukaryotic expression vector of pIRES2-ZsGreen1-rBD-2 was successfully constructed. The level of rBD-2 mRNA in transfected cells was significantly higher than that in control groups through the real-time fluorescence relative quantitative PCR. CONCLUSION: The recombinant eukaryotic expression vector pIRES2-ZsGreen1-rBD-2 could be transfected into rat corneal epithelial cells, and exogenous rBD-2 gene could be transcripted into mRNA in

Development of expression vectors for Escherichia coli based on the pCR2 replicon

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Deb J K

2007-05-01

Full Text Available Abstract Background Recent developments in metabolic engineering and the need for expanded compatibility required for co-expression studies, underscore the importance of developing new plasmid vectors with properties such as stability and compatibility. Results We utilized the pCR2 replicon of Corynebacterium renale, which harbours multiple plasmids, for constructing a range of expression vectors. Different antibiotic-resistance markers were introduced and the vectors were found to be 100% stable over a large number of generations in the absence of selection pressure. Compatibility of this plasmid was studied with different Escherichia coli plasmid replicons viz. pMB1 and p15A. It was observed that pCR2 was able to coexist with these E.coli plasmids for 60 generations in the absence of selection pressure. Soluble intracellular production was checked by expressing GFP under the lac promoter in an expression plasmid pCR2GFP. Also high level production of human IFNγ was obtained by cloning the h-IFNγ under a T7 promoter in the expression plasmid pCR2-IFNγ and using a dual plasmid heat shock system for expression. Repeated sub-culturing in the absence of selection pressure for six days did not lead to any fall in the production levels post induction, for both GFP and h-IFNγ, demonstrating that pCR2 is a useful plasmid in terms of stability and compatibility. Conclusion We have constructed a series of expression vectors based on the pCR2 replicon and demonstrated its high stability and sustained expression capacity, in the absence of selection pressure which will make it an efficient tool for metabolic engineering and co-expression studies, as well as for scale up of expression.

阴道毛滴虫TvRab11C基因的克隆及原核表达%Cloning and prokaryotic expression of TvRab11C gene of Trichomonas vaginalis

Institute of Scientific and Technical Information of China (English)

刘畅; 丁鹤; 宫鹏涛; 李建华; 李淑红; 李赫; 张国才; 张西臣

2012-01-01

目的 克隆并原核表达阴道毛滴虫TvRab11C(G3 Ras-related protein Rab11C)基因.方法 利用PCR技术扩增阴道毛滴虫TvRab1 1C基因,与原核表达载体pET-28a连接,构建重组原核表达质粒pET-28a-TvRab1 1C,转化大肠杆菌BL21(DE3),IPTG诱导表达,SDS-PAGE分析表达产物的可溶性,Western blot分析表达产物的反应原性.结果 重组原核表达质粒pET-28a-TvRab11C经双酶切及测序证明构建正确;表达的重组蛋白相对分子质量约为30000,主要以包涵体形式存在,可被抗阴道毛滴虫多克隆抗体识别.结论 成功克隆了阴道毛滴虫TvRab11C基因,并在E.coli BL21 (DE3)中获得了表达,为进一步研究TvRas基因和G蛋白与阴道毛滴虫寄生能力和致病性的关系奠定了基础.%Objective To clone the TvRabllC gene of Trichomonas vaginalis and express in prokaryotic cells. Methods The TvRabllC gene was amplified by PCR from T. vaginalis and inserted into prokaryotic expression vector pET-28a. The constructed recombinant plasmid pET-28a-TvRabllC was transformed to E. coli BL21 (DE3) and induced by IPTG. The expressed product was analyzed for solubility by SDS-PAGE and for reactogenicity by Western blot. Results Both restriction analysis and sequencing proved that recombinant plasmid pET-28a-TvRabllC was constructed correctly. The expressed recombinant protein,with a relative molecular mass of about 30 000,mainly existed in a form of inclusion body,and was recognized by polyclonal antibody against T. vaginalis. Conclusion The TvRas gene of T. vaginalis was successfully cloned and expressed in E. coli BL21 (DE3),which laid a foundation of further study on relationship of TvRas gene and protein to the parasitic ability and pathogenicity of T. vaginalis.

Ectopic expression of the erythrocyte band 3 anion exchange protein, using a new avian retrovirus vector

DEFF Research Database (Denmark)

Fuerstenberg, S; Beug, H; Introna, M

1990-01-01

into protein. Using the Escherichia coli beta-galactosidase gene cloned into the vector as a test construct, expression of enzyme activity could be detected in 90 to 95% of transfected target cells and in 80 to 85% of subsequently infected cells. In addition, a cDNA encoding the avian erythrocyte band 3 anion...... exchange protein has been expressed from the vector in both chicken embryo fibroblasts and QT6 cells and appears to function as an active, plasma membrane-based anion transporter. The ectopic expression of band 3 protein provides a visual marker for vector function in these cells....

Prokaryotic Expression of Truncate F Protein Gene of Newcastle Disease Virus%截短NDV F蛋白的原核表达

Institute of Scientific and Technical Information of China (English)

王杰; 王宇鹏; 刘振格; 杨鸣发; 林红丽; 田斌; 侯喜林

2013-01-01

根据NDV LaSota株F基因已知的抗原表位，对F蛋白进行分段表达。应用RT-PCR方法分段扩增F基因，并将其克隆到pET30a（+）原核表达载体上，得到重组质粒pET30-F780和pET30-F760，将质粒导入BL21（ED3）感受态中，经IPTG诱导表达。表达的重组蛋白通过SDS-PAGE和Westem-blotting方法进行鉴定。表达的两段蛋白大小约为31.1 kDa和27.9 kDa，与预期的蛋白分子量大小相符。Western blot分析表明重组蛋白可以和NDV抗体发生特异性反应。成功构建了原核表达质粒pET-F780和pET-F760，并获得了高效表达，通过Western blot分析表明重组蛋白具有良好的免疫反应性。%According to the epitopes of F gene of NDV LaSota strain,the F gene truncated two fragments were expressed in E.coli BL21 (ED3)strain. The two truncate F gene amplified by RT-PCR were inserted into pET30 (+),a prokaryotic expression vector. The recombinant plasmid pET30-F780 and pET30-F760 were transformed into BL21(ED3)competent cells. SDS-PAGE and Western-blotting screened the recombinant proteins induced by IPTG in E.coli. The size of the recombinant proteins were 31.1 kDa and 27.9 kDa,which were also consistent with those expected. Western-blotting showed that F780 and F760 were of immunogenicity.The recombinant plasmids were constructed,called pET-F780 and pET-F760,which were expressed the corresponding proteins with better immunoreactivity.

Recombinant vesicular stomatitis virus vaccine vectors expressing filovirus glycoproteins lack neurovirulence in nonhuman primates.

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Chad E Mire

Full Text Available The filoviruses, Marburg virus and Ebola virus, cause severe hemorrhagic fever with high mortality in humans and nonhuman primates. Among the most promising filovirus vaccines under development is a system based on recombinant vesicular stomatitis virus (rVSV that expresses an individual filovirus glycoprotein (GP in place of the VSV glycoprotein (G. The main concern with all replication-competent vaccines, including the rVSV filovirus GP vectors, is their safety. To address this concern, we performed a neurovirulence study using 21 cynomolgus macaques where the vaccines were administered intrathalamically. Seven animals received a rVSV vector expressing the Zaire ebolavirus (ZEBOV GP; seven animals received a rVSV vector expressing the Lake Victoria marburgvirus (MARV GP; three animals received rVSV-wild type (wt vector, and four animals received vehicle control. Two of three animals given rVSV-wt showed severe neurological symptoms whereas animals receiving vehicle control, rVSV-ZEBOV-GP, or rVSV-MARV-GP did not develop these symptoms. Histological analysis revealed major lesions in neural tissues of all three rVSV-wt animals; however, no significant lesions were observed in any animals from the filovirus vaccine or vehicle control groups. These data strongly suggest that rVSV filovirus GP vaccine vectors lack the neurovirulence properties associated with the rVSV-wt parent vector and support their further development as a vaccine platform for human use.

Multicistronic lentiviral vectors containing the FMDV 2A cleavage factor demonstrate robust expression of encoded genes at limiting MOI

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Margison Geoffrey P

2006-03-01

Full Text Available Abstract Background A number of gene therapy applications would benefit from vectors capable of expressing multiple genes. In this study we explored the feasibility and efficiency of expressing two or three transgenes in HIV-1 based lentiviral vector. Bicistronic and tricistronic self-inactivating lentiviral vectors were constructed employing the internal ribosomal entry site (IRES sequence of encephalomyocarditis virus (EMCV and/or foot-and-mouth disease virus (FMDV cleavage factor 2A. We employed enhanced green fluorescent protein (eGFP, O6-methylguanine-DNA-methyltransferase (MGMT, and homeobox transcription factor HOXB4 as model genes and their expression was detected by appropriate methods including fluorescence microscopy, flow cytometry, immunocytochemistry, biochemical assay, and western blotting. Results All the multigene vectors produced high titer virus and were able to simultaneously express two or three transgenes in transduced cells. However, the level of expression of individual transgenes varied depending on: the transgene itself; its position within the construct; the total number of transgenes expressed; the strategy used for multigene expression and the average copy number of pro-viral insertions. Notably, at limiting MOI, the expression of eGFP in a bicistronic vector based on 2A was ~4 times greater than that of an IRES based vector. Conclusion The small and efficient 2A sequence can be used alone or in combination with an IRES for the construction of multicistronic lentiviral vectors which can express encoded transgenes at functionally relevant levels in cells containing an average of one pro-viral insert.

[Optimization of prokaryotic expression conditions of Leptospira interrogans trigeminy genus-specific protein antigen based on surface response analysis].

Science.gov (United States)

Wang, Jiang; Luo, Dongjiao; Sun, Aihua; Yan, Jie

2008-07-01

Lipoproteins LipL32 and LipL21 and transmembrane protein OMPL1 have been confirmed as the superficial genus-specific antigens of Leptospira interrogans, which can be used as antigens for developing a universal genetic engineering vaccine. In order to obtain high expression of an artificial fusion gene lipL32/1-lipL21-ompL1/2, we optimized prokaryotic expression conditions. We used surface response analysis based on the central composite design to optimize culture conditions of a new antigen protein by recombinant Escherichia coli DE3.The culture conditions included initial pH, induction start time, post-induction time, Isopropyl beta-D-thiogalactopyranoside (IPTG) concentration, and temperature. The maximal production of antigen protein was 37.78 mg/l. The optimal culture conditions for high recombinant fusion protein was determined: initial pH 7.9, induction start time 2.5 h, a post-induction time of 5.38 h, 0.20 mM IPTG, and a post-induction temperature of 31 degrees C. Surface response analysis based on CCD increased the target production. This statistical method reduced the number of experiments required for optimization and enabled rapid identification and integration of the key culture condition parameters for optimizing recombinant protein expression.

Horizontal transfer, not duplication, drives the expansion of protein families in prokaryotes.

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Todd J Treangen

2011-01-01

Full Text Available Gene duplication followed by neo- or sub-functionalization deeply impacts the evolution of protein families and is regarded as the main source of adaptive functional novelty in eukaryotes. While there is ample evidence of adaptive gene duplication in prokaryotes, it is not clear whether duplication outweighs the contribution of horizontal gene transfer in the expansion of protein families. We analyzed closely related prokaryote strains or species with small genomes (Helicobacter, Neisseria, Streptococcus, Sulfolobus, average-sized genomes (Bacillus, Enterobacteriaceae, and large genomes (Pseudomonas, Bradyrhizobiaceae to untangle the effects of duplication and horizontal transfer. After removing the effects of transposable elements and phages, we show that the vast majority of expansions of protein families are due to transfer, even among large genomes. Transferred genes--xenologs--persist longer in prokaryotic lineages possibly due to a higher/longer adaptive role. On the other hand, duplicated genes--paralogs--are expressed more, and, when persistent, they evolve slower. This suggests that gene transfer and gene duplication have very different roles in shaping the evolution of biological systems: transfer allows the acquisition of new functions and duplication leads to higher gene dosage. Accordingly, we show that paralogs share most protein-protein interactions and genetic regulators, whereas xenologs share very few of them. Prokaryotes invented most of life's biochemical diversity. Therefore, the study of the evolution of biology systems should explicitly account for the predominant role of horizontal gene transfer in the diversification of protein families.

The human ankyrin 1 promoter insulator sustains gene expression in a β-globin lentiviral vector in hematopoietic stem cells

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Zulema Romero

Full Text Available Lentiviral vectors designed for the treatment of the hemoglobinopathies require the inclusion of regulatory and strong enhancer elements to achieve sufficient expression of the β-globin transgene. Despite the inclusion of these elements, the efficacy of these vectors may be limited by transgene silencing due to the genomic environment surrounding the integration site. Barrier insulators can be used to give more consistent expression and resist silencing even with lower vector copies. Here, the barrier activity of an insulator element from the human ankyrin-1 gene was analyzed in a lentiviral vector carrying an antisickling human β-globin gene. Inclusion of a single copy of the Ankyrin insulator did not affect viral titer, and improved the consistency of expression from the vector in murine erythroleukemia cells. The presence of the Ankyrin insulator element did not change transgene expression in human hematopoietic cells in short-term erythroid culture or in vivo in primary murine transplants. However, analysis in secondary recipients showed that the lentiviral vector with the Ankyrin element preserved transgene expression, whereas expression from the vector lacking the Ankyrin insulator decreased in secondary recipients. These studies demonstrate that the Ankyrin insulator may improve long-term β-globin expression in hematopoietic stem cells for gene therapy of hemoglobinopathies.

Recombinant vectors construction for cellobiohydrolase encoding gene constitutive expression

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Leontina GURGU

2012-12-01

Full Text Available Cellobiohydrolases (EC 3.2.1.91 are important exo enzymes involved in cellulose hydrolysis alongside endoglucanases (EC 3.2.1.4 and β-glucosidases (EC 3.2.1.21. Heterologous cellobiohydrolase gene expression under constitutive promoter control using Saccharomyces cerevisiae as host system is of great importance for a successful SSF process. From this point of view, the main objective of the work was to use Yeplac181 expression vector as a recipient for cellobiohdrolase - cbhB encoding gene expression under the control of the actin promoter, in Saccharomyces cerevisiae. Two hybridvectors, YEplac-Actp and YEplac-Actp-CbhB, were generated usingEscherichia coli XLI Blue for the cloning experiments. Constitutive cbhB gene expression was checked by proteine gel electrophoresis (SDS-PAGE after insertion of these constructs into Saccharomyces cerevisiae.

Cloning, expression and purification of d-tagatose 3-epimerase gene from Escherichia coli JM109.

Science.gov (United States)

He, Xiaoliang; Zhou, Xiaohui; Yang, Zi; Xu, Le; Yu, Yuxiu; Jia, Lingling; Li, Guoqing

2015-10-01

An unknown d-tagatose 3-epimerase (DTE) containing a IoIE domain was identified and cloned from Escherichia coli. This gene was subcloned into the prokaryotic expression vector pET-15b, and induced by IPTG in E. coli BL21 expression system. Through His-select gel column purification and fast-protein liquid chromatography, highly purified and stable DTE protein was produced. The molecular weight of the DTE protein was estimated to be 29.8kDa. The latest 83 DTE sequences from public database were selected and analyzed by molecular clustering, multi-sequence alignment. DTEs were roughly divided into five categories. Copyright © 2015 Elsevier Inc. All rights reserved.

Classification of e-government documents based on cooperative expression of word vectors

Science.gov (United States)

Fu, Qianqian; Liu, Hao; Wei, Zhiqiang

2017-03-01

The effective document classification is a powerful technique to deal with the huge amount of e-government documents automatically instead of accomplishing them manually. The word-to-vector (word2vec) model, which converts semantic word into low-dimensional vectors, could be successfully employed to classify the e-government documents. In this paper, we propose the cooperative expressions of word vector (Co-word-vector), whose multi-granularity of integration explores the possibility of modeling documents in the semantic space. Meanwhile, we also aim to improve the weighted continuous bag of words model based on word2vec model and distributed representation of topic-words based on LDA model. Furthermore, combining the two levels of word representation, performance result shows that our proposed method on the e-government document classification outperform than the traditional method.

Suppression of leaky expression of adenovirus genes by insertion of microRNA-targeted sequences in the replication-incompetent adenovirus vector genome

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Kahori Shimizu

2014-01-01

Full Text Available Leaky expression of adenovirus (Ad genes occurs following transduction with a conventional replication-incompetent Ad vector, leading to an induction of cellular immunity against Ad proteins and Ad protein-induced toxicity, especially in the late phase following administration. To suppress the leaky expression of Ad genes, we developed novel Ad vectors by incorporating four tandem copies of sequences with perfect complementarity to miR-122a or miR-142-3p into the 3′-untranslated region (UTR of the E2A, E4, or pIX gene, which were mainly expressed from the Ad vector genome after transduction. These Ad vectors easily grew to high titers comparable to those of a conventional Ad vector in conventional 293 cells. The leaky expression of these Ad genes in mouse organs was significantly suppressed by 2- to 100-fold, compared with a conventional Ad vector, by insertion of the miRNA-targeted sequences. Notably, the Ad vector carrying the miR-122a–targeted sequences into the 3′-UTR of the E4 gene expressed higher and longer-term transgene expression and more than 20-fold lower levels of all the Ad early and late genes examined in the liver than a conventional Ad vector. miR-122a–mediated suppression of the E4 gene expression in the liver significantly reduced the hepatotoxicity which an Ad vector causes via both adaptive and non-adaptive immune responses.

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

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Xi-jun CHEN

2011-12-01

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

High-Throughput Agrobacterium-mediated Transformation of Medicago Truncatula in Comparison to Two Expression Vectors

International Nuclear Information System (INIS)

Sultana, T.; Deeba, F.; Naqvi, S. M. S.

2016-01-01

Legumes have been turbulent to efficient Agrobacterium-mediated transformation for a long time. The selection of Medicago truncatula as a model legume plant for molecular analysis resulted in the development of efficient Agrobacterium-mediated transformation protocols. In current study, M. truncatula transformed plants expressing OsRGLP1 were obtained through GATEWAY technology using pGOsRGLP1 (pH7WG2.0=OsRGLP1). The transformation efficiency of this vector was compared with expression vector from pCAMBIA series over-expressing same gene (pCOsRGLP1). A lower number of explants generated hygromycin resistant plantlet for instance, 18.3 with pGOsRGLP1 vector as compared to 35.5 percent with pCOsRGLP1 vector. Transformation efficiency of PCR positive plants generated was 9.4 percent for pGOsRGLP1 while 21.6 percent for pCOsRGLP1. Furthermore 24.4 percent of explants generated antibiotic resistant plantlet on 20 mgl/sup -1/ of hygromycin which was higher than on 15 mgl/sup -1/ of hygromycin such as 12.2 percent. T/sub 1/ progeny analysis indicated that the transgene was inherited in Mendelian manner. The functionally active status of transgene was monitored by high level of Superoxide dismutase (SOD) activity in transformed progeny. (author)

Facial Expression Recognition using Multiclass Ensemble Least-Square Support Vector Machine

Science.gov (United States)

Lawi, Armin; Sya'Rani Machrizzandi, M.

2018-03-01

Facial expression is one of behavior characteristics of human-being. The use of biometrics technology system with facial expression characteristics makes it possible to recognize a person’s mood or emotion. The basic components of facial expression analysis system are face detection, face image extraction, facial classification and facial expressions recognition. This paper uses Principal Component Analysis (PCA) algorithm to extract facial features with expression parameters, i.e., happy, sad, neutral, angry, fear, and disgusted. Then Multiclass Ensemble Least-Squares Support Vector Machine (MELS-SVM) is used for the classification process of facial expression. The result of MELS-SVM model obtained from our 185 different expression images of 10 persons showed high accuracy level of 99.998% using RBF kernel.

Capturing prokaryotic dark matter genomes.

Science.gov (United States)

Gasc, Cyrielle; Ribière, Céline; Parisot, Nicolas; Beugnot, Réjane; Defois, Clémence; Petit-Biderre, Corinne; Boucher, Delphine; Peyretaillade, Eric; Peyret, Pierre

2015-12-01

Prokaryotes are the most diverse and abundant cellular life forms on Earth. Most of them, identified by indirect molecular approaches, belong to microbial dark matter. The advent of metagenomic and single-cell genomic approaches has highlighted the metabolic capabilities of numerous members of this dark matter through genome reconstruction. Thus, linking functions back to the species has revolutionized our understanding of how ecosystem function is sustained by the microbial world. This review will present discoveries acquired through the illumination of prokaryotic dark matter genomes by these innovative approaches. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Low-Dose Gene Therapy for Murine PKU Using Episomal Naked DNA Vectors Expressing PAH from Its Endogenous Liver Promoter

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Hiu Man Grisch-Chan

2017-06-01

Full Text Available Limited duration of transgene expression, insertional mutagenesis, and size limitations for transgene cassettes pose challenges and risk factors for many gene therapy vectors. Here, we report on physiological expression of liver phenylalanine hydroxylase (PAH by delivery of naked DNA/minicircle (MC-based vectors for correction of homozygous enu2 mice, a model of human phenylketonuria (PKU. Because MC vectors lack a defined size limit, we constructed a MC vector expressing a codon-optimized murine Pah cDNA that includes a truncated intron and is under the transcriptional control of a 3.6-kb native Pah promoter/enhancer sequence. This vector, delivered via hydrodynamic injection, yielded therapeutic liver PAH activity and sustained correction of blood phenylalanine comparable to viral or synthetic liver promoters. Therapeutic efficacy was seen with vector copy numbers of 95% loss of vector genomes and PAH activity in liver, demonstrating that MC vectors had not integrated into the liver genome. In conclusion, MC vectors, which do not have a defined size-limitation, offer a favorable safety profile for hepatic gene therapy due to their non-integration in combination with native promoters.

Adenovirus vector expressing keratinocyte growth factor using CAG promoter impairs pulmonary function of mice with elastase-induced emphysema.

Science.gov (United States)

Oki, Hiroshi; Yazawa, Takuya; Baba, Yasuko; Kanegae, Yumi; Sato, Hanako; Sakamoto, Seiko; Goto, Takahisa; Saito, Izumu; Kurahashi, Kiyoyasu

2017-07-01

Pulmonary emphysema impairs quality of life and increases mortality. It has previously been shown that administration of adenovirus vector expressing murine keratinocyte growth factor (KGF) before elastase instillation prevents pulmonary emphysema in mice. We therefore hypothesized that therapeutic administration of KGF would restore damage to lungs caused by elastase instillation and thus improve pulmonary function in an animal model. KGF expressing adenovirus vector, which prevented bleomycin-induced pulmonary fibrosis in a previous study, was constructed. Adenovirus vector (1.0 × 10 9 plaque-forming units) was administered intratracheally one week after administration of elastase into mouse lungs. One week after administration of KGF-vector, exercise tolerance testing and blood gas analysis were performed, after which the lungs were removed under deep anesthesia. KGF-positive pneumocytes were more numerous, surfactant protein secretion in the airspace greater and mean linear intercept of lungs shorter in animals that had received KGF than in control animals. Unexpectedly, however, arterial blood oxygenation was worse in the KGF group and maximum running speed, an indicator of exercise capacity, had not improved after KGF in mice with elastase-induced emphysema, indicating that KGF-expressing adenovirus vector impaired pulmonary function in these mice. Notably, vector lacking KGF-expression unit did not induce such impairment, implying that the KGF expression unit itself may cause the damage to alveolar cells. Possible involvement of the CAG promoter used for KGF expression in impairing pulmonary function is discussed. © 2017 The Societies and John Wiley & Sons Australia, Ltd.

A proposed genus boundary for the prokaryotes based on genomic insights.

Science.gov (United States)

Qin, Qi-Long; Xie, Bin-Bin; Zhang, Xi-Ying; Chen, Xiu-Lan; Zhou, Bai-Cheng; Zhou, Jizhong; Oren, Aharon; Zhang, Yu-Zhong

2014-06-01

Genomic information has already been applied to prokaryotic species definition and classification. However, the contribution of the genome sequence to prokaryotic genus delimitation has been less studied. To gain insights into genus definition for the prokaryotes, we attempted to reveal the genus-level genomic differences in the current prokaryotic classification system and to delineate the boundary of a genus on the basis of genomic information. The average nucleotide sequence identity between two genomes can be used for prokaryotic species delineation, but it is not suitable for genus demarcation. We used the percentage of conserved proteins (POCP) between two strains to estimate their evolutionary and phenotypic distance. A comprehensive genomic survey indicated that the POCP can serve as a robust genomic index for establishing the genus boundary for prokaryotic groups. Basically, two species belonging to the same genus would share at least half of their proteins. In a specific lineage, the genus and family/order ranks showed slight or no overlap in terms of POCP values. A prokaryotic genus can be defined as a group of species with all pairwise POCP values higher than 50%. Integration of whole-genome data into the current taxonomy system can provide comprehensive information for prokaryotic genus definition and delimitation. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

The establishment of Saccharomyces boulardii surface display system using a single expression vector.

Science.gov (United States)

Wang, Tiantian; Sun, Hui; Zhang, Jie; Liu, Qing; Wang, Longjiang; Chen, Peipei; Wang, Fangkun; Li, Hongmei; Xiao, Yihong; Zhao, Xiaomin

2014-03-01

In the present study, an a-agglutinin-based Saccharomyces boulardii surface display system was successfully established using a single expression vector. Based on the two protein co-expression vector pSP-G1 built by Partow et al., a S. boulardii surface display vector-pSDSb containing all the display elements was constructed. The display results of heterologous proteins were confirmed by successfully displaying enhanced green fluorescent protein (EGFP) and chicken Eimeria tenella Microneme-2 proteins (EtMic2) on the S. boulardii cell surface. The DNA sequence of AGA1 gene from S. boulardii (SbAGA1) was determined and used as the cell wall anchor partner. This is the first time heterologous proteins have been displayed on the cell surface of S. boulardii. Because S. boulardii is probiotic and eukaryotic, its surface display system would be very valuable, particularly in the development of a live vaccine against various pathogenic organisms especially eukaryotic pathogens such as protistan parasites. Copyright © 2013 Elsevier Inc. All rights reserved.

RNA polymerase II mediated transcription from the polymerase III promoters in short hairpin RNA expression vector

International Nuclear Information System (INIS)

Rumi, Mohammad; Ishihara, Shunji; Aziz, Monowar; Kazumori, Hideaki; Ishimura, Norihisa; Yuki, Takafumi; Kadota, Chikara; Kadowaki, Yasunori; Kinoshita, Yoshikazu

2006-01-01

RNA polymerase III promoters of human ribonuclease P RNA component H1, human U6, and mouse U6 small nuclear RNA genes are commonly used in short hairpin RNA (shRNA) expression vectors due their precise initiation and termination sites. During transient transfection of shRNA vectors, we observed that H1 or U6 promoters also express longer transcripts enough to express several reporter genes including firefly luciferase, green fluorescent protein EGFP, and red fluorescent protein JRed. Expression of such longer transcripts was augmented by upstream RNA polymerase II enhancers and completely inhibited by downstream polyA signal sequences. Moreover, the transcription of firefly luciferase from human H1 promoter was sensitive to RNA polymerase II inhibitor α-amanitin. Our findings suggest that commonly used polymerase III promoters in shRNA vectors are also prone to RNA polymerase II mediated transcription, which may have negative impacts on their targeted use

UMG Lenti: novel lentiviral vectors for efficient transgene- and reporter gene expression in human early hematopoietic progenitors.

Directory of Open Access Journals (Sweden)

Emanuela Chiarella

Full Text Available Lentiviral vectors are widely used to investigate the biological properties of regulatory proteins and/or of leukaemia-associated oncogenes by stably enforcing their expression in hematopoietic stem and progenitor cells. In these studies it is critical to be able to monitor and/or sort the infected cells, typically via fluorescent proteins encoded by the modified viral genome. The most popular strategy to ensure co-expression of transgene and reporter gene is to insert between these cDNAs an IRES element, thus generating bi-cistronic mRNAs whose transcription is driven by a single promoter. However, while the product of the gene located upstream of the IRES is generally abundantly expressed, the translation of the downstream cDNA (typically encoding the reporter protein is often inconsistent, which hinders the detection and the isolation of transduced cells. To overcome these limitations, we developed novel lentiviral dual-promoter vectors (named UMG-LV5 and -LV6 where transgene expression is driven by the potent UBC promoter and that of the reporter protein, EGFP, by the minimal regulatory element of the WASP gene. These vectors, harboring two distinct transgenes, were tested in a variety of human haematopoietic cell lines as well as in primary human CD34+ cells in comparison with the FUIGW vector that contains the expression cassette UBC-transgene-IRES-EGFP. In these experiments both UMG-LV5 and UMG-LV6 yielded moderately lower transgene expression than FUIGW, but dramatically higher levels of EGFP, thereby allowing the easy distinction between transduced and non-transduced cells. An additional construct was produced, in which the cDNA encoding the reporter protein is upstream, and the transgene downstream of the IRES sequence. This vector, named UMG-LV11, proved able to promote abundant expression of both transgene product and EGFP in all cells tested. The UMG-LVs represent therefore useful vectors for gene transfer-based studies in

UMG Lenti: novel lentiviral vectors for efficient transgene- and reporter gene expression in human early hematopoietic progenitors.

Science.gov (United States)

Chiarella, Emanuela; Carrà, Giovanna; Scicchitano, Stefania; Codispoti, Bruna; Mega, Tiziana; Lupia, Michela; Pelaggi, Daniela; Marafioti, Maria G; Aloisio, Annamaria; Giordano, Marco; Nappo, Giovanna; Spoleti, Cristina B; Grillone, Teresa; Giovannone, Emilia D; Spina, Raffaella; Bernaudo, Francesca; Moore, Malcolm A S; Bond, Heather M; Mesuraca, Maria; Morrone, Giovanni

2014-01-01

Lentiviral vectors are widely used to investigate the biological properties of regulatory proteins and/or of leukaemia-associated oncogenes by stably enforcing their expression in hematopoietic stem and progenitor cells. In these studies it is critical to be able to monitor and/or sort the infected cells, typically via fluorescent proteins encoded by the modified viral genome. The most popular strategy to ensure co-expression of transgene and reporter gene is to insert between these cDNAs an IRES element, thus generating bi-cistronic mRNAs whose transcription is driven by a single promoter. However, while the product of the gene located upstream of the IRES is generally abundantly expressed, the translation of the downstream cDNA (typically encoding the reporter protein) is often inconsistent, which hinders the detection and the isolation of transduced cells. To overcome these limitations, we developed novel lentiviral dual-promoter vectors (named UMG-LV5 and -LV6) where transgene expression is driven by the potent UBC promoter and that of the reporter protein, EGFP, by the minimal regulatory element of the WASP gene. These vectors, harboring two distinct transgenes, were tested in a variety of human haematopoietic cell lines as well as in primary human CD34+ cells in comparison with the FUIGW vector that contains the expression cassette UBC-transgene-IRES-EGFP. In these experiments both UMG-LV5 and UMG-LV6 yielded moderately lower transgene expression than FUIGW, but dramatically higher levels of EGFP, thereby allowing the easy distinction between transduced and non-transduced cells. An additional construct was produced, in which the cDNA encoding the reporter protein is upstream, and the transgene downstream of the IRES sequence. This vector, named UMG-LV11, proved able to promote abundant expression of both transgene product and EGFP in all cells tested. The UMG-LVs represent therefore useful vectors for gene transfer-based studies in hematopoietic stem and

A simple and robust vector-based shRNA expression system used for RNA interference.

Science.gov (United States)

Wang, Xue-jun; Li, Ying; Huang, Hai; Zhang, Xiu-juan; Xie, Pei-wen; Hu, Wei; Li, Dan-dan; Wang, Sheng-qi

2013-01-01

RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) has become a powerful genetic tool for conducting functional studies. Previously, vector-based shRNA-expression strategies capable of inducing RNAi in viable cells have been developed, however, these vector systems have some disadvantages, either because they were error-prone or cost prohibitive. In this report we described the development of a simple, robust shRNA expression system utilizing 1 long oligonucleotide or 2 short oligonucleotides for half the cost of conventional shRNA construction methods and with a >95% cloning success rate. The shRNA loop sequence and stem structure were also compared and carefully selected for better RNAi efficiency. Furthermore, an easier strategy was developed based on isocaudomers which permit rapid combination of the most efficient promoter-shRNA cassettes. Finally, using this method, the conservative target sites for hepatitis B virus (HBV) knockdown were systemically screened and HBV antigen expression shown to be successfully suppressed in the presence of connected multiple shRNAs both in vitro and in vivo. This novel design describes an inexpensive and effective way to clone and express single or multiple shRNAs from the same vector with the capacity for potent and effective silencing of target genes.

A Versatile System for USER Cloning-Based Assembly of Expression Vectors for Mammalian Cell Engineering

DEFF Research Database (Denmark)

Lund, Anne Mathilde; Kildegaard, Helene Faustrup; Petersen, Maja Borup Kjær

2014-01-01

, in addition the system is fully extendable by other users. The vector system is designed to facilitate high-throughput genome-scale studies of mammalian cells, such as the newly sequenced CHO cell lines, through the ability to rapidly generate high-fidelity assembly of customizable gene expression vectors....

Viral Diversity Threshold for Adaptive Immunity in Prokaryotes

Science.gov (United States)

Weinberger, Ariel D.; Wolf, Yuri I.; Lobkovsky, Alexander E.; Gilmore, Michael S.; Koonin, Eugene V.

2012-01-01

ABSTRACT Bacteria and archaea face continual onslaughts of rapidly diversifying viruses and plasmids. Many prokaryotes maintain adaptive immune systems known as clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (Cas). CRISPR-Cas systems are genomic sensors that serially acquire viral and plasmid DNA fragments (spacers) that are utilized to target and cleave matching viral and plasmid DNA in subsequent genomic invasions, offering critical immunological memory. Only 50% of sequenced bacteria possess CRISPR-Cas immunity, in contrast to over 90% of sequenced archaea. To probe why half of bacteria lack CRISPR-Cas immunity, we combined comparative genomics and mathematical modeling. Analysis of hundreds of diverse prokaryotic genomes shows that CRISPR-Cas systems are substantially more prevalent in thermophiles than in mesophiles. With sequenced bacteria disproportionately mesophilic and sequenced archaea mostly thermophilic, the presence of CRISPR-Cas appears to depend more on environmental temperature than on bacterial-archaeal taxonomy. Mutation rates are typically severalfold higher in mesophilic prokaryotes than in thermophilic prokaryotes. To quantitatively test whether accelerated viral mutation leads microbes to lose CRISPR-Cas systems, we developed a stochastic model of virus-CRISPR coevolution. The model competes CRISPR-Cas-positive (CRISPR-Cas+) prokaryotes against CRISPR-Cas-negative (CRISPR-Cas−) prokaryotes, continually weighing the antiviral benefits conferred by CRISPR-Cas immunity against its fitness costs. Tracking this cost-benefit analysis across parameter space reveals viral mutation rate thresholds beyond which CRISPR-Cas cannot provide sufficient immunity and is purged from host populations. These results offer a simple, testable viral diversity hypothesis to explain why mesophilic bacteria disproportionately lack CRISPR-Cas immunity. More generally, fundamental limits on the adaptability of biological

Mycobacterium tuberculosis HspX/EsxS Fusion Protein: Gene Cloning, Protein Expression, and Purification in Escherichia coli.

Science.gov (United States)

Khademi, Farzad; Yousefi-Avarvand, Arshid; Derakhshan, Mohammad; Meshkat, Zahra; Tafaghodi, Mohsen; Ghazvini, Kiarash; Aryan, Ehsan; Sankian, Mojtaba

2017-10-01

The purpose of this study was to clone, express, and purify a novel multidomain fusion protein of Micobacterium tuberculosis (Mtb) in a prokaryotic system. An hspX/esxS gene construct was synthesized and ligated into a pGH plasmid, E. coli TOP10 cells were transformed, and the vector was purified. The vector containing the construct and pET-21b (+) plasmid were digested with the same enzymes and the construct was ligated into pET-21b (+). The accuracy of cloning was confirmed by colony PCR and sequencing. E. coli BL21 cells were transformed with the pET-21b (+)/hspX/esxS expression vector and protein expression was evaluated. Finally, the expressed fusion protein was purified on a Ni-IDA column and verified by SDS-PAGE and western blotting. The hspX/esxS gene construct was inserted into pET-21b (+) and recombinant protein expression was induced with IPTG in E. coli BL21 cells. Various concentrations of IPTG were tested to determine the optimum concentration for expression induction. The recombinant protein was expressed in insoluble inclusion bodies. Three molar guanidine HCl was used to solubilize the insoluble protein. An HspX/EsxS Mtb fusion protein was expressed in E. coli and the recombinant protein was purified. After immunological analysis, the HspX/EsxS fusion protein might be an anti-tuberculosis vaccine candidate in future clinical trial studies.

Prokaryotic Argonautes - variations on the RNA interference theme

Science.gov (United States)

van der Oost, John; Swarts, Daan C.; Jore, Matthijs M.

2014-01-01

The discovery of RNA interference (RNAi) has been a major scientific breakthrough. This RNA-guided RNA interference system plays a crucial role in a wide range of regulatory and defense mechanisms in eukaryotes. The key enzyme of the RNAi system is Argonaute (Ago), an endo-ribonuclease that uses a small RNA guide molecule to specifically target a complementary RNA transcript. Two functional classes of eukaryotic Ago have been described: catalytically active Ago that cleaves RNA targets complementary to its guide, and inactive Ago that uses its guide to bind target RNA to down-regulate translation efficiency. A recent comparative genomics study has revealed that Argonaute-like proteins are also encoded by prokaryotic genomes. Interestingly, there is a lot of variation among these prokaryotic Argonaute (pAgo) proteins with respect to domain architecture: some resemble the eukaryotic Ago (long pAgo) containing a complete or disrupted catalytic site, while others are truncated versions (short pAgo) that generally contain an incomplete catalytic site. Prokaryotic Agos with an incomplete catalytic site often co-occur with (predicted) nucleases. Based on this diversity, and on the fact that homologs of other RNAi-related protein components (such as Dicer nucleases) have never been identified in prokaryotes, it has been predicted that variations on the eukaryotic RNAi theme may occur in prokaryotes. PMID:28357239

Cytomegalovirus vector expressing RAE-1γ induces enhanced anti-tumor capacity of murine CD8+ T cells.

Science.gov (United States)

Tršan, Tihana; Vuković, Kristina; Filipović, Petra; Brizić, Ana Lesac; Lemmermann, Niels A W; Schober, Kilian; Busch, Dirk H; Britt, William J; Messerle, Martin; Krmpotić, Astrid; Jonjić, Stipan

2017-08-01

Designing CD8 + T-cell vaccines, which would provide protection against tumors is still considered a great challenge in immunotherapy. Here we show the robust potential of cytomegalovirus (CMV) vector expressing the NKG2D ligand RAE-1γ as CD8 + T cell-based vaccine against malignant tumors. Immunization with the CMV vector expressing RAE-1γ, delayed tumor growth or even provided complete protection against tumor challenge in both prophylactic and therapeutic settings. Moreover, a potent tumor control in mice vaccinated with this vector can be further enhanced by blocking the immune checkpoints TIGIT and PD-1. CMV vector expressing RAE-1γ potentiated expansion of KLRG1 + CD8 + T cells with enhanced effector properties. This vaccination was even more efficient in neonatal mice, resulting in the expansion and long-term maintenance of epitope-specific CD8 + T cells conferring robust resistance against tumor challenge. Our data show that immunomodulation of CD8 + T-cell responses promoted by herpesvirus expressing a ligand for NKG2D receptor can provide a powerful platform for the prevention and treatment of CD8 + T-cell sensitive tumors. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Rotations with Rodrigues' vector

International Nuclear Information System (INIS)

Pina, E

2011-01-01

The rotational dynamics was studied from the point of view of Rodrigues' vector. This vector is defined here by its connection with other forms of parametrization of the rotation matrix. The rotation matrix was expressed in terms of this vector. The angular velocity was computed using the components of Rodrigues' vector as coordinates. It appears to be a fundamental matrix that is used to express the components of the angular velocity, the rotation matrix and the angular momentum vector. The Hamiltonian formalism of rotational dynamics in terms of this vector uses the same matrix. The quantization of the rotational dynamics is performed with simple rules if one uses Rodrigues' vector and similar formal expressions for the quantum operators that mimic the Hamiltonian classical dynamics.

Effect of nitrate addition on prokaryotic diversity and the activity of sulfate-reducing prokaryotes in high-temperature oil production systems

DEFF Research Database (Denmark)

Gittel, Antje; Wieczorek, Adam; Sørensen, Ketil

Adding nitrate to injection water is a possible strategy to control the activity of sulfate-reducing prokaryotes (SRP) in oil production system. To assess the effects of nitrate addition, prokaryotic diversity (Bacteria, Archaea, SRP) and SRP activity were studied in the production waters......-treated site was additionally supported by demonstrating their potential activity at 58°C, indicating that the troublesome SRP were pipeline-derived. Consistent with the low frequency of SRP in the clone libraries, no activity could be shown for samples from the nitrate-treated system suggesting that SRP were...... inhibited by nitrate addition. Visualization and quantification of the identified troublesome prokaryotes and potential competitors using the CARD-FISH technique will be performed on production water from both sites....

The Epigenomic Landscape of Prokaryotes.

Directory of Open Access Journals (Sweden)

Matthew J Blow

2016-02-01

Full Text Available DNA methylation acts in concert with restriction enzymes to protect the integrity of prokaryotic genomes. Studies in a limited number of organisms suggest that methylation also contributes to prokaryotic genome regulation, but the prevalence and properties of such non-restriction-associated methylation systems remain poorly understood. Here, we used single molecule, real-time sequencing to map DNA modifications including m6A, m4C, and m5C across the genomes of 230 diverse bacterial and archaeal species. We observed DNA methylation in nearly all (93% organisms examined, and identified a total of 834 distinct reproducibly methylated motifs. This data enabled annotation of the DNA binding specificities of 620 DNA Methyltransferases (MTases, doubling known specificities for previously hard to study Type I, IIG and III MTases, and revealing their extraordinary diversity. Strikingly, 48% of organisms harbor active Type II MTases with no apparent cognate restriction enzyme. These active 'orphan' MTases are present in diverse bacterial and archaeal phyla and show motif specificities and methylation patterns consistent with functions in gene regulation and DNA replication. Our results reveal the pervasive presence of DNA methylation throughout the prokaryotic kingdoms, as well as the diversity of sequence specificities and potential functions of DNA methylation systems.

Prokaryotic DNA segregation by an actin-like filament

DEFF Research Database (Denmark)

Møller-Jensen, Jakob; Bugge Jensen, Rasmus; Löwe, Jan

2002-01-01

The mechanisms responsible for prokaryotic DNA segregation are largely unknown. The partitioning locus (par) encoded by the Escherichia coli plasmid R1 actively segregates its replicon to daughter cells. We show here that the ParM ATPase encoded by par forms dynamic actin-like filaments with prop...... point for ParM polymerization. Hence, we provide evidence for a simple prokaryotic analogue of the eukaryotic mitotic spindle apparatus.......The mechanisms responsible for prokaryotic DNA segregation are largely unknown. The partitioning locus (par) encoded by the Escherichia coli plasmid R1 actively segregates its replicon to daughter cells. We show here that the ParM ATPase encoded by par forms dynamic actin-like filaments...

Elimination of contaminating cap genes in AAV vector virions reduces immune responses and improves transgene expression in a canine gene therapy model.

Science.gov (United States)

Wang, Z; Halbert, C L; Lee, D; Butts, T; Tapscott, S J; Storb, R; Miller, A D

2014-04-01

Animal and human gene therapy studies utilizing AAV vectors have shown that immune responses to AAV capsid proteins can severely limit transgene expression. The main source of capsid antigen is that associated with the AAV vectors, which can be reduced by stringent vector purification. A second source of AAV capsid proteins is that expressed from cap genes aberrantly packaged into AAV virions during vector production. This antigen source can be eliminated by the use of a cap gene that is too large to be incorporated into an AAV capsid, such as a cap gene containing a large intron (captron gene). Here, we investigated the effects of elimination of cap gene transfer and of vector purification by CsCl gradient centrifugation on AAV vector immunogenicity and expression following intramuscular injection in dogs. We found that both approaches reduced vector immunogenicity and that combining the two produced the lowest immune responses and highest transgene expression. This combined approach enabled the use of a relatively mild immunosuppressive regimen to promote robust micro-dystrophin gene expression in Duchenne muscular dystrophy-affected dogs. Our study shows the importance of minimizing AAV cap gene impurities and indicates that this improvement in AAV vector production may benefit human applications.

Modulating ectopic gene expression levels by using retroviral vectors equipped with synthetic promoters

OpenAIRE

Ferreira, Joshua P.; Peacock, Ryan W. S.; Lawhorn, Ingrid E. B.; Wang, Clifford L.

2011-01-01

The human cytomegalovirus and elongation factor 1α promoters are constitutive promoters commonly employed by mammalian expression vectors. These promoters generally produce high levels of expression in many types of cells and tissues. To generate a library of synthetic promoters capable of generating a range of low, intermediate, and high expression levels, the TATA and CAAT box elements of these promoters were mutated. Other promoter variants were also generated by random mutagenesis. Evalua...

Phase 2 clinical trial of a recombinant adeno-associated viral vector expressing α1-antitrypsin: interim results.

LENUS (Irish Health Repository)

Flotte, Terence R

2011-10-01

Recombinant adeno-associated virus (rAAV) vectors offer promise for the gene therapy of α(1)-antitrypsin (AAT) deficiency. In our prior trial, an rAAV vector expressing human AAT (rAAV1-CB-hAAT) provided sustained, vector-derived AAT expression for >1 year. In the current phase 2 clinical trial, this same vector, produced by a herpes simplex virus complementation method, was administered to nine AAT-deficient individuals by intramuscular injection at doses of 6.0×10(11), 1.9×10(12), and 6.0×10(12) vector genomes\\/kg (n=3 subjects\\/dose). Vector-derived expression of normal (M-type) AAT in serum was dose dependent, peaked on day 30, and persisted for at least 90 days. Vector administration was well tolerated, with only mild injection site reactions and no serious adverse events. Serum creatine kinase was transiently elevated on day 30 in five of six subjects in the two higher dose groups and normalized by day 45. As expected, all subjects developed anti-AAV antibodies and interferon-γ enzyme-linked immunospot responses to AAV peptides, and no subjects developed antibodies to AAT. One subject in the mid-dose group developed T cell responses to a single AAT peptide unassociated with any clinical effects. Muscle biopsies obtained on day 90 showed strong immunostaining for AAT and moderate to marked inflammatory cell infiltrates composed primarily of CD3-reactive T lymphocytes that were primarily of the CD8(+) subtype. These results support the feasibility and safety of AAV gene therapy for AAT deficiency, and indicate that serum levels of vector-derived normal human AAT >20 μg\\/ml can be achieved. However, further improvements in the design or delivery of rAAV-AAT vectors will be required to achieve therapeutic target serum AAT concentrations.

A simple and robust vector-based shRNA expression system used for RNA interference.

Directory of Open Access Journals (Sweden)

Xue-jun Wang

Full Text Available BACKGROUND: RNA interference (RNAi mediated by small interfering RNAs (siRNAs or short hairpin RNAs (shRNAs has become a powerful genetic tool for conducting functional studies. Previously, vector-based shRNA-expression strategies capable of inducing RNAi in viable cells have been developed, however, these vector systems have some disadvantages, either because they were error-prone or cost prohibitive. RESULTS: In this report we described the development of a simple, robust shRNA expression system utilizing 1 long oligonucleotide or 2 short oligonucleotides for half the cost of conventional shRNA construction methods and with a >95% cloning success rate. The shRNA loop sequence and stem structure were also compared and carefully selected for better RNAi efficiency. Furthermore, an easier strategy was developed based on isocaudomers which permit rapid combination of the most efficient promoter-shRNA cassettes. Finally, using this method, the conservative target sites for hepatitis B virus (HBV knockdown were systemically screened and HBV antigen expression shown to be successfully suppressed in the presence of connected multiple shRNAs both in vitro and in vivo. CONCLUSION: This novel design describes an inexpensive and effective way to clone and express single or multiple shRNAs from the same vector with the capacity for potent and effective silencing of target genes.

[Construction of the lentiviral expression vector for anti-p185(erbB2) mouse/human chimeric antibody].

Science.gov (United States)

Liu, Fang; Li, Li; Zhang, Wei; Wang, Qi

2013-04-01

This research was to construct the lentiviral expression vector for anti- p185(erbB2) mouse/human chimeric antibody and to determine the expression of the chimeric antibody gene in 293T cells transfected with this vector. The genes (vL and vH) coding light and heavy chain of variable regions of anti-p185(erbB2) mAb and the constant regions of human IgG1 (kappa and gamma1) were cloned with PCR method. The target genes were assembled by three-primers PCR method to obtain the chimeric light chain (L) and the chimeric heavy chain (H). Both chains inserted into the down stream and upper stream of IRES gene of the plasmid pVAX1/IRES respectively. We digested the plasmid pVAX1/ H-IRES-L with endoenzyme and subcloned H-IRES-L into the lentiviral vector pWPI. The enzyme digestion and sequence analysis showed that the lentiviral expression vector pWPI/H-IRES-L was constructed correctly. Then, it was transfected into 293T cells and after 48h, GFP protein expression in 293T cells were detected by fluorescent microscope and the chimeric antibody expression was detected by RT-PCR and direct ELISA. The results showed that after 293T cells were transfected with recombination plasmid, both light and heavy chains of the chimeric antibody genes could express together. The chimeric antibody expressed could bind to p185(erbB2) specifically. This research may lay a sound foundation for further study of anti-p185(erbB2) engineered antibody.

Novel viral vectors utilizing intron splice-switching to activate genome rescue, expression and replication in targeted cells

Directory of Open Access Journals (Sweden)

El Andaloussi Samir

2011-05-01

Full Text Available Abstract Background The outcome of virus infection depends from the precise coordination of viral gene expression and genome replication. The ability to control and regulate these processes is therefore important for analysis of infection process. Viruses are also useful tools in bio- and gene technology; they can efficiently kill cancer cells and trigger immune responses to tumors. However, the methods for constructing tissue- or cell-type specific viruses typically suffer from low target-cell specificity and a high risk of reversion. Therefore novel and universal methods of regulation of viral infection are also important for therapeutic application of virus-based systems. Methods Aberrantly spliced introns were introduced into crucial gene-expression units of adenovirus vector and alphavirus DNA/RNA layered vectors and their effects on the viral gene expression, replication and/or the release of infectious genomes were studied in cell culture. Transfection of the cells with splice-switching oligonucleotides was used to correct the introduced functional defect(s. Results It was demonstrated that viral gene expression, replication and/or the release of infectious genomes can be blocked by the introduction of aberrantly spliced introns. The insertion of such an intron into an adenovirus vector reduced the expression of the targeted gene more than fifty-fold. A similar insertion into an alphavirus DNA/RNA layered vector had a less dramatic effect; here, only the release of the infectious transcript was suppressed but not the subsequent replication and spread of the virus. However the insertion of two aberrantly spliced introns resulted in an over one hundred-fold reduction in the infectivity of the DNA/RNA layered vector. Furthermore, in both systems the observed effects could be reverted by the delivery of splice-switching oligonucleotide(s, which corrected the splicing defects. Conclusions Splice-switch technology, originally developed for

Differential transgene expression in brain cells in vivo and in vitro from AAV-2 vectors with small transcriptional control units

International Nuclear Information System (INIS)

Kuegler, S.; Lingor, P.; Schoell, U.; Zolotukhin, S.; Baehr, M.

2003-01-01

Adeno-associated- (AAV) based vectors are promising tools for gene therapy applications in several organs, including the brain, but are limited by their small genome size. Two short promoters, the human synapsin 1 gene promoter (hSYN) and the murine cytomegalovirus immediate early promoter (mCMV), were evaluated in bicistronic AAV-2 vectors for their expression profiles in cultured primary brain cells and in the rat brain. Whereas transgene expression from the hSYN promoter was exclusively neuronal, the murine CMV promoter targeted expression mainly to astrocytes in vitro and showed weak transgene expression in vivo in retinal and cortical neurons, but strong expression in thalamic neurons. We propose that neuron specific transgene expression in combination with enhanced transgene capacity will further substantially improve AAV based vector technology

Expression of heterologous genes from an IRES translational cassette in replication-competent murine leukemia virus vectors

DEFF Research Database (Denmark)

Jespersen, T.; Duch, M.; Carrasco, M.L.

1999-01-01

of spliced env mRNA for the SL3-3 derived vector relative to the Akv derived vectors, seemingly contributing to its low replication capacity. The EGFP expressing Akv-MLV was genetically stable for multiple rounds of infection; marker-cassette deletion revertants appeared after several replication rounds...

Insect cell transformation vectors that support high level expression and promoter assessment in insect cell culture

Science.gov (United States)

A somatic transformation vector, pDP9, was constructed that provides a simplified means of producing permanently transformed cultured insect cells that support high levels of protein expression of foreign genes. The pDP9 plasmid vector incorporates DNA sequences from the Junonia coenia densovirus th...

Construction of expression vectors for metabolic engineering of the vanillin-producing actinomycete Amycolatopsis sp. ATCC 39116.

Science.gov (United States)

Fleige, Christian; Steinbüchel, Alexander

2014-01-01

Amycolatopsis sp. ATCC 39116 is able to synthesize the important flavoring agent vanillin from cheap natural substrates. The bacterium is therefore of great interest for the industry and used for the fermentative production of vanillin. In order to improve the production of natural vanillin with Amycolatopsis sp. ATCC 39116, the strain has been genetically engineered to optimize the metabolic flux towards the desired product. Extensive metabolic engineering was hitherto hampered, due to the lack of genetic tools like functional promoters and expression vectors. In this study, we report the establishment of a plasmid-based gene expression system for Amycolatopsis sp. ATCC 39116 that allows a further manipulation of the genotype. Four new Escherichia coli-Amycolatopsis shuttle vectors harboring different promoter elements were constructed, and the functionality of these regulatory elements was proven by the expression of the reporter gene gusA, encoding a β-glucuronidase. Glucuronidase activity was detected in all plasmid-harboring strains, and remarkable differences in the expression strength of the reporter gene depending on the used promoter were observed. The new expression vectors will promote the further genetic engineering of Amycolatopsis sp. ATCC 39116 to get insight into the metabolic network and to improve the strain for a more efficient industrial use.

Biological and immunogenic properties of rabies virus glycoprotein expressed by canine herpesvirus vector.

Science.gov (United States)

Xuan, X; Tuchiya, K; Sato, I; Nishikawa, Y; Onoderaz, Y; Takashima, Y; Yamamoto, A; Katsumata, A; Iwata, A; Ueda, S; Mikami, T; Otsuka, H

1998-01-01

In order to evaluate whether canine herpesvirus (CHV) could be used as a live vector for the expression of heterologous immunogenes, we constructed a recombinant canine herpesvirus (CHV) expressing glycoprotein (G protein) of rabies virus (RV). The gene of G protein was inserted within the thymidine kinase gene of CHV YP11mu strain under the control of the human cytomegalovirus immediate early promoter. The G protein expressed by the recombinant CHV was processed and transported to the cell surface as in RV infected cells, and showed the same biological activities such as low pH dependent cell fusion and hemadsorption. The antigenic authenticity of the recombinant G protein was confirmed by a panel of monoclonal antibodies specific for G protein. Dogs inoculated intransally with the recombinant CHV produced higher titres of virus neutralizing antibodies against RV than those inoculated with a commercial, inactivated rabies vaccine. These results suggest that the CHV recombinant expressing G protein can be used as a vaccine to control canine rabies and that CHV may be useful as a vector to develop live recombinant against other infectious diseases in dogs.

Construction and Development of a Cardiac Tissue-Specific and Hypoxia-Inducible Expression Vector

Directory of Open Access Journals (Sweden)

Shahrooz Ghaderi

2018-03-01

Full Text Available Purpose: Cardiovascular gene therapy is a sophisticated approach, thanks to the safety of vectors, stable transgene expression, delivery method, and different layers of the heart. To date, numerous expression vectors have been introduced in biotechnology and biopharmacy industries in relation to genetic manipulation. Despite the rapid growth of these modalities, they must be intelligently designed, addressing the cardiac-specific transgene expression and less side effects. Herein, we conducted a pilot project aiming to design a cardiac-specific hypoxia-inducible expression cassette. Methods: We explored a new approach to design an expression cassette containing cardiac specific enhancer, hypoxia response elements (HRE, cardiac specific promoter, internal ribosome entry site (IRES, and beta globin poly A sequence to elicit specific and inducible expression of the gene of interest. Enhanced green fluorescent protein (eGFP was sub-cloned by BglII and NotI into the cassette. The specificity and inducible expression of the cassette was determined in both mouse myoblast C2C12 and mammary glandular tumor 4T1 as ‘twin’ cells. eGFP expression was evaluated by immunofluorescence microscope and flow cytometry at 520 nm emission peak. Results: Our data revealed that the designed expression cassette provided tissue specific and hypoxia inducible (O2<1% transgene expression. Conclusion: It is suggested that cardiac-specific enhancer combined with cardiac-specific promoter are efficient for myoblast specific gene expression. As well, this is for the first time that HRE are derived from three well known hypoxia-regulated promoters. Therefore, there is no longer need to overlap PCR process for one repeated sequence just in one promoter.

Engineering and Validation of a Vector for Concomitant Expression of Rare Transfer RNA (tRNA and HIV-1 nef Genes in Escherichia coli.

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Siti Aisyah Mualif

Full Text Available Relative ease in handling and manipulation of Escherichia coli strains make them primary candidate to express proteins heterologously. Overexpression of heterologous genes that contain codons infrequently used by E. coli is related with difficulties such as mRNA instability, early termination of transcription and/or translation, deletions and/or misincorporation, and cell growth inhibition. These codon bias -associated problems are addressed by co-expressing ColE1-compatible, rare tRNA expressing helper plasmids. However, this approach has inadequacies, which we have addressed by engineering an expression vector that concomitantly expresses the heterologous protein of interest, and rare tRNA genes in E. coli. The expression vector contains three (argU, ileY, leuW rare tRNA genes and a useful multiple cloning site for easy in-frame cloning. To maintain the overall size of the parental plasmid vector, the rare tRNA genes replaced the non-essential DNA segments in the vector. The cloned gene is expressed under the control of T7 promoter and resulting recombinant protein has a C-terminal 6His tag for IMAC-mediated purification. We have evaluated the usefulness of this expression vector by expressing three HIV-1 genes namely HIV-1 p27 (nef, HIV-1 p24 (ca, and HIV-1 vif in NiCo21(DE3 E.coli and demonstrated the advantages of using expression vector that concomitantly expresses rare tRNA and heterologous genes.

Hypoxia-inducible bidirectional shRNA expression vector delivery using PEI/chitosan-TBA copolymers for colorectal Cancer gene therapy.

Science.gov (United States)

Javan, Bita; Atyabi, Fatemeh; Shahbazi, Majid

2018-04-12

This investigation was conducted to construct a hypoxia/colorectal dual-specific bidirectional short hairpin RNA (shRNA) expression vector and to transfect it into the colon cancer cell line HT-29 with PEI/chitosan-TBA nanoparticles for the simultaneous knock down of β-catenin and Bcl-2 under hypoxia. To construct a pRNA-bipHRE-CEA vector, the carcinoma embryonic antigen (CEA) promoter designed in two directions and the vascular endothelial growth factor (VEGF) enhancer were inserted between two promoters for hypoxic cancer specific gene expression. To confirm the therapeutic effect of the dual-specific vector, β-catenin and Bcl-2 shRNAs were inserted downstream of each promoter. The physicochemical properties, the cytotoxicity, and the transfection efficiency of these PEI/chitosan-TBA nanoparticles were investigated. In addition, the antitumor effects of the designed vector on the expression of β-catenin and Bcl-2, cell cycle distribution, and apoptosis were investigated in vitro. The silencing effect of the hypoxia-response shRNA expression vector was relatively low (18%-25%) under normoxia, whereas it was significantly increased to approximately 50%-60% in the HT-29 cell line. Moreover, the cancer cells showed significant G0/G1 arrest and increased apoptosis due to gene silencing under hypoxia. Furthermore, MTS assay, fluorescence microscopy images, and flow cytometry analyses confirmed that the PEI/chitosan-TBA blend system provided effective transfection with low cytotoxicity. This novel hypoxia-responsive shRNA expression vector may be useful for RNA interference (RNAi)-based cancer gene therapy in hypoxic colorectal tumors. Moreover, the PEI/chitosan-TBA copolymer might be a promising gene carrier for use in gene transfer in vivo. Copyright © 2017. Published by Elsevier Inc.

Insights into structural variations and genome rearrangements in prokaryotic genomes.

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Periwal, Vinita; Scaria, Vinod

2015-01-01

Structural variations (SVs) are genomic rearrangements that affect fairly large fragments of DNA. Most of the SVs such as inversions, deletions and translocations have been largely studied in context of genetic diseases in eukaryotes. However, recent studies demonstrate that genome rearrangements can also have profound impact on prokaryotic genomes, leading to altered cell phenotype. In contrast to single-nucleotide variations, SVs provide a much deeper insight into organization of bacterial genomes at a much better resolution. SVs can confer change in gene copy number, creation of new genes, altered gene expression and many other functional consequences. High-throughput technologies have now made it possible to explore SVs at a much refined resolution in bacterial genomes. Through this review, we aim to highlight the importance of the less explored field of SVs in prokaryotic genomes and their impact. We also discuss its potential applicability in the emerging fields of synthetic biology and genome engineering where targeted SVs could serve to create sophisticated and accurate genome editing. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Primer design for a prokaryotic differential display RT-PCR.

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Fislage, R; Berceanu, M; Humboldt, Y; Wendt, M; Oberender, H

1997-05-01

We have developed a primer set for a prokaryotic differential display of mRNA in the Enterobacteriaceae group. Each combination of ten 10mer and ten 11mer primers generates up to 85 bands from total Escherichia coli RNA, thus covering expressed sequences of a complete bacterial genome. Due to the lack of polyadenylation in prokaryotic RNA the type T11VN anchored oligonucleotides for the reverse transcriptase reaction had to be replaced with respect to the original method described by Liang and Pardee [ Science , 257, 967-971 (1992)]. Therefore, the sequences of both the 10mer and the new 11mer oligonucleotides were determined by a statistical evaluation of species-specific coding regions extracted from the EMBL database. The 11mer primers used for reverse transcription were selected for localization in the 3'-region of the bacterial RNA. The 10mer primers preferentially bind to the 5'-end of the RNA. None of the primers show homology to rRNA or other abundant small RNA species. Randomly sampled cDNA bands were checked for their bacterial origin either by re-amplification, cloning and sequencing or by re-amplification and direct sequencing with 10mer and 11mer primers after asymmetric PCR.

Novel Strategy to Control Transgene Expression Mediated by a Sendai Virus-Based Vector Using a Nonstructural C Protein and Endogenous MicroRNAs.

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Masayuki Sano

Full Text Available Tissue-specific control of gene expression is an invaluable tool for studying various biological processes and medical applications. Efficient regulatory systems have been utilized to control transgene expression in various types of DNA viral or integrating viral vectors. However, existing regulatory systems are difficult to transfer into negative-strand RNA virus vector platforms because of significant differences in their transcriptional machineries. In this study, we developed a novel strategy for regulating transgene expression mediated by a cytoplasmic RNA vector based on a replication-defective and persistent Sendai virus (SeVdp. Because of the capacity of Sendai virus (SeV nonstructural C proteins to specifically inhibit viral RNA synthesis, overexpression of C protein significantly reduced transgene expression mediated by SeVdp vectors. We found that SeV C overexpression concomitantly reduced SeVdp mRNA levels and genomic RNA synthesis. To control C expression, target sequences for an endogenous microRNA were incorporated into the 3' untranslated region of the C genes. Incorporation of target sequences for miR-21 into the SeVdp vector restored transgene expression in HeLa cells by decreasing C expression. Furthermore, the SeVdp vector containing target sequences for let-7a enabled cell-specific control of transgene expression in human fibroblasts and induced pluripotent stem cells. Our findings demonstrate that SeV C can be used as an effective regulator for controlling transgene expression. This strategy will contribute to efficient and less toxic SeVdp-mediated gene transfer in various biological applications.

[Prokaryotic expression and immunogenicity analysis of the chimeric HBcAg containing APP beta cleavage site peptide and Aβ(1-15);].

Science.gov (United States)

Feng, Gai-feng; Wang, Jun-yang; Jin, Hui; Wang, Wei-xi; Qian, Yi-hua; Yang, Wei-na; Wang, Quan-ying; Yang, Guang-xiao

2011-11-01

To construct the recombinant prokaryotic expression plasmid pET/c-ABCSP-Aβ(15-c);, and evaluate the immunogenicity of the fusion protein expressed in E.coli. The gene fragment HBc88-144 was amplified by PCR and subcloned to pUC19. The APP beta cleavage site peptide(ABCSP) and Aβ(1-15); gene(ABCSP-Aβ(15);) was amplified by PCR and inserted downstream of HBc1-71 in pGEMEX/c1-71. After restriction enzyme digestion, c1-17-ABCSP-Aβ(15); were connected with HBc88-144, yielding the recombinant gene c-ABCSP-Aβ(15-c);. c-ABCSP-Aβ(15-c); gene was subcloned into pET-28a(+).The fusion protein expressed in transformed E.coli BL21 was induced with IPTG and analyzed by SDS-PAGE. The virus-like particles (VLP) formed by fusion protein was observed with Transmission Electron Microscope (TEM). 4 Kunming (KM) mice received intraperitoneal injection (i.p) of fusion protein VLP. The antibody was detected by indirect ELISA. The recombinant gene was confirmed by restriction enzyme digestion and DNA sequencing. After IPTG induction, fusion protein was expressed and mainly existed in the sediment of the bacterial lysate. The expression level was 40% of all the proteins in the sediment. The fusion protein could form VLP. After 5 times of immunization, the titer of anti-ABCSP and anti-Aβantibody in sera of KM mice reached up to 1:5 000 and 1:10 000 respectively, while the anti-HBc antibody was undetectable. Recombinant c-ABCSP-Aβ(15-c); gene can be expressed in E.coli. The expressed protein could form VLP and has a strong immunogenicity. This study lays the foundation for the study of AD genetic engineering vaccine.

Construction of a CD147 Lentiviral Expression Vector and Establishment of Its Stably Transfected A549 Cell Line

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Shaoxing YANG

2012-12-01

Full Text Available Background and objective CD147, a type of transmembrane glycoprotein embedded on the surface of tumor cells, can promote tumor invasion and metastasis. This aim of this study is to construct a CD147 lentiviral expression vector, establish its stably transfected A549 cell line, and observe the effect of CD147 on MMP-9 proliferation as well as on the invasive ability of human lung adenocarcinoma cells. Methods Full-length CD147 gene was amplified by real-time polymerase chain reaction (RT-PCR, inserted into a pEGFP vector to construct pEGFP-CD147 and pEGFP vectors, and then transfected into 293FT cells to precede the lentivirus equipment package. Subsequently, we collected the lentivirus venom to infect the A549 cells and establish a stable, overexpressed cell line named A549-CD147. The mRNA expression of MMP-9 was examined by RT-PCR. The proliferation and invasive ability of the human lung cancer cells before and after transfection were examined by the CCK-8 and Transwell methods. Results A CD147 lentiviral expression vector (pEGFP-CD147 was successfully constructed by restrictive enzyme digestion and plasmid sequencing. RT-PCR and Western blot analyses revealed increased mRNA and protein expression of CD147 gene in cells transfected with pEGFP-CD147 compared with the control groups. Therefore, the A549-CD147 cell line was successfully established through the experiment. The mRNA expression of MMP-9 also significantly increased after the upregulation of CD147 expression. Meanwhile, CCK-8 and Transwell assays indicated that the proliferation and invasive ability significantly increased in the A549-CD147 cells. Conclusion A lentiviral CD147 expression vector and its A549 cell line (A549-CD14 were successfully constructed. CD147 overexpression upregulated the protein expression of MMP-9, and strengthened the proliferation and invasive ability of human lung adenocarcinoma cells.

Efficient genome editing in hematopoietic stem cells with helper-dependent Ad5/35 vectors expressing site-specific endonucleases under microRNA regulation

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Kamola Saydaminova

Full Text Available Genome editing with site-specific endonucleases has implications for basic biomedical research as well as for gene therapy. We generated helper-dependent, capsid-modified adenovirus (HD-Ad5/35 vectors for zinc-finger nuclease (ZFN– or transcription activator-like effector nuclease (TALEN–mediated genome editing in human CD34+ hematopoietic stem cells (HSCs from mobilized adult donors. The production of these vectors required that ZFN and TALEN expression in HD-Ad5/35 producer 293-Cre cells was suppressed. To do this, we developed a microRNA (miRNA-based system for regulation of gene expression based on miRNA expression profiling of 293-Cre and CD34+ cells. Using miR-183-5p and miR-218-5p based regulation of transgene gene expression, we first produced an HD-Ad5/35 vector expressing a ZFN specific to the HIV coreceptor gene ccr5. We demonstrated that HD-Ad5/35.ZFNmiR vector conferred ccr5 knock out in primitive HSC (i.e., long-term culture initiating cells and NOD/SCID repopulating cells. The ccr5 gene disruption frequency achieved in engrafted HSCs found in the bone marrow of transplanted mice is clinically relevant for HIV therapy considering that these cells can give rise to multiple lineages, including all the lineages that represent targets and reservoirs for HIV. We produced a second HD-Ad5/35 vector expressing a TALEN targeting the DNase hypersensitivity region 2 (HS2 within the globin locus control region. This vector has potential for targeted gene correction in hemoglobinopathies. The miRNA regulated HD-Ad5/35 vector platform for expression of site-specific endonucleases has numerous advantages over currently used vectors as a tool for genome engineering of HSCs for therapeutic purposes.

Interactions Between Prokaryotes and Dissolved Organic Matter in Marine Waters

DEFF Research Database (Denmark)

Traving, Sachia Jo

organic bound carbon equal in size to atmospheric carbon dioxide. Prokaryotes mediate the fate of a large part of marine DOM, which is their principal source of energy and substrate. However, a large fraction is also left behind in the water column persisting for millennia, and prokaryotes may hold...... the key to understanding the mechanisms controlling the cycling of DOM within marine waters. In the thesis presented here, the aim was to investigate the activity and composition of prokaryotes to determine their functional role in DOM utilization. The thesis incorporates a range of study systems...

Light-controlled motility in prokaryotes and the problem of directional light perception.

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Wilde, Annegret; Mullineaux, Conrad W

2017-11-01

The natural light environment is important to many prokaryotes. Most obviously, phototrophic prokaryotes need to acclimate their photosynthetic apparatus to the prevailing light conditions, and such acclimation is frequently complemented by motility to enable cells to relocate in search of more favorable illumination conditions. Non-phototrophic prokaryotes may also seek to avoid light at damaging intensities and wavelengths, and many prokaryotes with diverse lifestyles could potentially exploit light signals as a rich source of information about their surroundings and a cue for acclimation and behavior. Here we discuss our current understanding of the ways in which bacteria can perceive the intensity, wavelength and direction of illumination, and the signal transduction networks that link light perception to the control of motile behavior. We discuss the problems of light perception at the prokaryotic scale, and the challenge of directional light perception in small bacterial cells. We explain the peculiarities and the common features of light-controlled motility systems in prokaryotes as diverse as cyanobacteria, purple photosynthetic bacteria, chemoheterotrophic bacteria and haloarchaea. © FEMS 2017.

The Role of Prokaryotes in Sediment Carbon Cycling

DEFF Research Database (Denmark)

Piil, Kristoffer

in the sediment. In particular, the work has focused on estimating how rapidly amino acids derived from plankton are degraded and replaced by amino acids from prokaryotes and how extensive this reworking of amino acids is in surface sediments. Another part of my work has focused on establishing reliable estimates...... of cell specific amino acid and muramic acid concentrations in sediment bacteria. Such estimates are important tools when studying the reworking of amino acids by bacteria and the preservation of bacterial cell walls. In addition, it has been an aim of the work to investigate how abundant endospores...... are in marine sediment and how dynamic the endospore population is, as very little is known about this compartment of the prokaryotic community. Prokaryotic reworking of amino acids was investigated by two independent methods. The first approach involved estimating the amount of amino acids produced...

Clustered regularly interspaced short palindromic repeats (CRISPRs): the hallmark of an ingenious antiviral defense mechanism in prokaryotes.

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Al-Attar, Sinan; Westra, Edze R; van der Oost, John; Brouns, Stan J J

2011-04-01

Many prokaryotes contain the recently discovered defense system against mobile genetic elements. This defense system contains a unique type of repetitive DNA stretches, termed Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs). CRISPRs consist of identical repeated DNA sequences (repeats), interspaced by highly variable sequences referred to as spacers. The spacers originate from either phages or plasmids and comprise the prokaryotes' 'immunological memory'. CRISPR-associated (cas) genes encode conserved proteins that together with CRISPRs make-up the CRISPR/Cas system, responsible for defending the prokaryotic cell against invaders. CRISPR-mediated resistance has been proposed to involve three stages: (i) CRISPR-Adaptation, the invader DNA is encountered by the CRISPR/Cas machinery and an invader-derived short DNA fragment is incorporated in the CRISPR array. (ii) CRISPR-Expression, the CRISPR array is transcribed and the transcript is processed by Cas proteins. (iii) CRISPR-Interference, the invaders' nucleic acid is recognized by complementarity to the crRNA and neutralized. An application of the CRISPR/Cas system is the immunization of industry-relevant prokaryotes (or eukaryotes) against mobile-genetic invasion. In addition, the high variability of the CRISPR spacer content can be exploited for phylogenetic and evolutionary studies. Despite impressive progress during the last couple of years, the elucidation of several fundamental details will be a major challenge in future research.

Differential response of marine flagellate communities to prokaryotic food quality

Science.gov (United States)

De Corte, D.; Paredes, G.; Sintes, E.; Herndl, G. J.

2016-02-01

Marine prokaryotes play a major role in the biogeochemical cycles. The main predators of prokaryotes are heterotrophic nanoflagellates (HNF). HNF are thus a major link connecting dissolved organic material through prokaryotic grazing to the higher trophic levels. However, little is known about the grazing specificity of HNF on specific prokaryotic taxa. Bacterial and archaeal microbes may have different nutritive values for the HNF communities, thus affecting growth rates and community composition of HNFs. In this study we investigated the influence of prey food quality on Cafeteria roenbergensis and on a natural HNF community isolated in the northern Adriatic Sea. Two Nitrosopumilus maritimus-related strains isolated from the northern Adriatic Sea (Nitrosopumilus adriaticus, Nitrosopumilus piranensis), two Nitrosococcus strains and two fast growing marine Bacteria (Pseudomonas marina and Marinobacter algicola) were fed to the HNFs. The two fast growing bacterial strains resulted in high growth rates of Cafeteria roenbergensis and the mixed HNF community, while the two Nitrosococcus strains did not. Cafeteria roenbergensis fed on N. adriaticus but it did not graze N. piranensis, suggesting that the subtle metabolic and physiological differences between these two closely related thaumarchaeal strains affect the grazing pressure to which they are exposed. Our study also indicates that prokaryotic community composition influences the composition of the HNF community.

Expression and purification of recombinant truncated human keratinocyte growth factor-1

International Nuclear Information System (INIS)

Deng Lin; Ma Jisheng; Liu Xiaoju; Wang Xiaojie; Li Xiaokun; Gong Shouliang; Wang Huiyan; Tian Haishan

2010-01-01

Objective: To construct the genetic engineering bacteria highly expressing 23 amino acids human keratinocyte growth factor-1 (rhKGF1 dest23 ) missing N terminal, and provide experimental data for development of new drug for treatment of oral mucositis after radiotherapy and chemotherapy. Methods: PCR was used to synthese 23 amino acids rhKGF1 dest23 missing N terminal and sumo gene fragments, and construct four kinds of recombinant prokaryotic expression vectors: pET22b-rhKGF1 dest23 , pET22b-sumo-rhKGF1 dest23 , pET3c-rhKGF1 dest23 and pET3c-sumo-rhKGF1 dest23 , then they were transformed into prokaryotic expression host bacteria: Rosetta (DE3) plysS, BL21 (DE3), BL21 (DE3) Star plysS, origima(DE3) and BL21AI, the best expression combination of plasmid and host strain of rhKGF1 dest23 protein was screened and purified by CM ion-exchange and heparin affinity chromatography and identified with Western blotting. Results: pET22b-rhKGF1 dest23 plasmid and the BL21AI host bacteria was the best combination of expression, after induced by IPTG and arabinose, the majority of recombinant protein was expressed in soluble form, accounting for about 12% of the total bacterial proteins. Its purity reached to more than 95% of the protein after two steps chromatography, then conformed with Western blotting. Conclusion: Human genetic engineering bacteria of KGF1 dest23 is successfully constructed and induced by IPTG and arabinose, then after CM weak cation exchange and heparin affinity chromatography, the purified rhKGF1 dest23 protein is obtained. (authors)

Three new shRNA expression vectors targeting the CYP3A4 coding sequence to inhibit its expression

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Siyun Xu

2014-10-01

Full Text Available RNA interference (RNAi is useful for selective gene silencing. Cytochrome P450 3A4 (CYP3A4, which metabolizes approximately 50% of drugs in clinical use, plays an important role in drug metabolism. In this study, we aimed to develop a short hairpin RNA (shRNA to modulate CYP3A4 expression. Three new shRNAs (S1, S2 and S3 were designed to target the coding sequence (CDS of CYP3A4, cloned into a shRNA expression vector, and tested in different cells. The mixture of three shRNAs produced optimal reduction (55% in CYP3A4 CDS-luciferase activity in both CHL and HEK293 cells. Endogenous CYP3A4 expression in HepG2 cells was decreased about 50% at both mRNA and protein level after transfection of the mixture of three shRNAs. In contrast, CYP3A5 gene expression was not altered by the shRNAs, supporting the selectivity of CYP3A4 shRNAs. In addition, HepG2 cells transfected with CYP3A4 shRNAs were less sensitive to Ginkgolic acids, whose toxic metabolites are produced by CYP3A4. These results demonstrate that vector-based shRNAs could modulate CYP3A4 expression in cells through their actions on CYP3A4 CDS, and CYP3A4 shRNAs may be utilized to define the role of CYP3A4 in drug metabolism and toxicity.

DNA Minicircle Technology Improves Purity of Adeno-associated Viral Vector Preparations

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Maria Schnödt

2016-01-01

Full Text Available Adeno-associated viral (AAV vectors are considered as one of the most promising delivery systems in human gene therapy. In addition, AAV vectors are frequently applied tools in preclinical and basic research. Despite this success, manufacturing pure AAV vector preparations remains a difficult task. While empty capsids can be removed from vector preparations owing to their lower density, state-of-the-art purification strategies as of yet failed to remove antibiotic resistance genes or other plasmid backbone sequences. Here, we report the development of minicircle (MC constructs to replace AAV vector and helper plasmids for production of both, single-stranded (ss and self-complementary (sc AAV vectors. As bacterial backbone sequences are removed during MC production, encapsidation of prokaryotic plasmid backbone sequences is avoided. This is of particular importance for scAAV vector preparations, which contained an unproportionally high amount of plasmid backbone sequences (up to 26.1% versus up to 2.9% (ssAAV. Replacing standard packaging plasmids by MC constructs not only allowed to reduce these contaminations below quantification limit, but in addition improved transduction efficiencies of scAAV preparations up to 30-fold. Thus, MC technology offers an easy to implement modification of standard AAV packaging protocols that significantly improves the quality of AAV vector preparations.

HDAd5/35++ Adenovirus Vector Expressing Anti-CRISPR Peptides Decreases CRISPR/Cas9 Toxicity in Human Hematopoietic Stem Cells

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Chang Li

2018-06-01

Full Text Available We generated helper-dependent HDAd5/35++ adenovirus vectors expressing CRISPR/Cas9 for potential hematopoietic stem cells (HSCs gene therapy of β-thalassemia and sickle cell disease through re-activation of fetal γ-globin expression (HDAd-globin-CRISPR. The process of CRISPR/Cas9 gene transfer using these vectors was not associated with death of human CD34+ cells and did not affect their in vitro expansion and erythroid differentiation. However, functional assays for primitive HSCs, e.g., multi-lineage progenitor colony formation and engraftment in irradiated NOD/Shi-scid/interleukin-2 receptor γ (IL-2Rγ null (NSG mice, revealed toxicity of HDAd-globin-CRISPR vectors related to the prolonged expression and activity of CRISPR/Cas9. To control the duration of CRISPR/Cas9 activity, we generated an HDAd5/35++ vector that expressed two anti-CRISPR (Acr peptides (AcrII4 and AcrII2 capable of binding to the CRISPR/Cas9 complex (HDAd-Acr. CD34+ cells that were sequentially infected with HDAd-CRISPR and HDAd-Acr engrafted at a significantly higher rate. Target site disruption frequencies in engrafted human cells were similar to those in pre-transplantation CD34+ cells, indicating that genome-edited primitive HSCs survived. In vitro differentiated HSCs isolated from transplanted mice demonstrated increased γ-globin expression as a result of genome editing. Our data indicate that the HDAd-Acr vector can be used as a tool to reduce HSC cytotoxicity of the CRISPR/Cas9 complex.

Suppression of inflammation by dexamethasone prolongs adenoviral vector-mediated transgene expression in the facial nucleus of the rat

NARCIS (Netherlands)

Hermens, W.T.J.M.C.; Verhaagen, J

1998-01-01

Adenoviral vector directed gene transfer to rat facial motoneurons occurs efficiently following intra-parenchymal injection of relatively high dosages (> or =10(7) pfu per injection) of a prototype first generation adenoviral vector. However, high level of transgene expression, as observed during

A lentiviral vector with expression controlled by E2F-1: A potential tool for the study and treatment of proliferative diseases

International Nuclear Information System (INIS)

Strauss, Bryan E.; Patricio, Juliana Rotelli; Vieira de Carvalho, Anna Carolina; Bajgelman, Marcio C.

2006-01-01

We have constructed a lentiviral vector with expression limited to cells presenting active E2F-1 protein, a potential advantage for gene therapy of proliferative diseases. For the FE2FLW vector, the promoter region of the human E2F-1 gene was utilized to drive expression of luciferase cDNA, included as a reporter of viral expression. Primary, immortalized, and transformed cells were transduced with the FE2FLW vector and cell cycle alterations were induced with serum starvation/replacement, contact inhibition or drug treatment, revealing cell cycle-dependent changes in reporter activity. Forced E2F-1 expression, but not E2F-2 or E2F-3, increased reporter activity, indicating a major role for this factor in controlling expression from the FE2FLW virus. We show the utility of this vector as a reporter of E2F-1 and proliferation-dependent cellular alterations upon cytotoxic/cytostatic treatment, such as the introduction of tumor suppressor genes. We propose that the FE2FLW vector may be a starting point for the development of gene therapy strategies for proliferative diseases, such as cancer or restinosis

[Construction of the eukaryotic recombinant vector and expression of the outer membrane protein LipL32 gene from Leptospira serovar Lai].

Science.gov (United States)

Huang, Bi; Bao, Lang; Zhong, Qi; Shang, Zheng-ling; Zhang, Hui-dong; Zhang, Ying

2008-02-01

To construct the eukaryotic experssion vector of LipL32 gene from Leptospira serovar Lai and express the recombinant plasmid in COS-7 cell. The LipL32 gene was amplified from Leptospira strain 017 genomic DNA by PCR and cloned into pcDNA3.1, through restriction nuclease enzyme digestion. Then the recombinant plasmid was transformed into E.coli DH5alpha. After identified by nuclease digestion, PCR and sequencing analysis, the recombinant vector was transfected into COS-7 cell with lipsome. The expression of the target gene was detected by RT-PCR and Western blot. The eukaryotic experssion vector pcDNA3.1-LipL32 was successfully constructed and stably expressed in COS-7 cell. The eukaryotic recombinant vector of outer membrane protein LipL32 gene from Leptospira serovar Lai can be expressed in mammalian cell, which provides an experimental basis for the application of the Leptospira DNA vaccine.

Study on construction of pEgr-hPTEN expression vector induced by irradiation and its anti-tumor effect in vitro

International Nuclear Information System (INIS)

Tian Mei; Jin Guanghui; Piao Chunji; Li Xiuyi; Liu Linlin

2003-01-01

Objective: To clone the cDNA of human tumor suppressor gene-PTEN, construct pEgr-hPTEN expression vector induced by irradiation and study its inhibitory effect on proliferation of malignant glioma cell line SHG-44 transfected steadily with pEgr-hPTEN after different doses of X-ray irradiation. Methods: A DNA fragment about 1200 bp, PTEN, was amplified from human placenta tissues by using RT-nested PCR and was cloned into pUCm-T vector after automatic sequencing, then the fragment was inserted into a vector pcD-NA3.1-Egr to construct an expression vector pEgr-hPTEN. pEgr-hPTEN was transfected into SHG-44 cells in vitro. Stably transfected cell line SHG-44-sPTEN was selected through G418. The inhibitor effect on SHG-44-sPTEN was observed after different doses of X-ray irradiation in vitro. Results: The PTEN cDNA has been cloned correctly and its expression vector pEgr-hPTEN was also constructed. Growth of SHG-44 cells was inhibited significantly by stable pEgr-hPTEN transfection combined with X-ray irradiation. With the increase of dose, the inhibitory effect was enhanced within 5 Gy. Conclusion: Human tumor suppressor gene-PTEN cDNA has been cloned and its expression vector has been constructed. The tumor was inhibited significantly by gene-radiotherapy in vitro. The result provides the theoretical and experimental basis for improvement of clinical radiotherapeutic effect on tumors

Top-Down Control of Diesel-Degrading Prokaryotic Communities.

Science.gov (United States)

Sauret, Caroline; Böttjer, Daniela; Talarmin, Agathe; Guigue, Catherine; Conan, Pascal; Pujo-Pay, Mireille; Ghiglione, Jean-François

2015-08-01

Biostimulation through the addition of inorganic nutrients has been the most widely practiced bioremediation strategy in oil-polluted marine waters. However, little attention has so far been paid to the microbial food web and the impact of top-down control that directly or indirectly influences the success of the bioremediation. We designed a mesocosm experiment using pre-filtered (diesel fuel. Prokaryotes, HNF and VLP abundances showed a predator-prey succession, with a co-development of HNF and VLP. In the polluted system, we observed a stronger impact of viral lysis on prokaryotic abundances than in the control. Analysis of the diversity revealed that a bloom of Vibrio sp. occurred in the polluted mesocosm. That bloom was rapidly followed by a less abundant and more even community of predation-resistant bacteria, including known hydrocarbon degraders such as Oleispira spp. and Methylophaga spp. and opportunistic bacteria such as Percisivirga spp., Roseobacter spp. and Phaeobacter spp. The shift in prokaryotic dominance in response to viral lysis provided clear evidence of the 'killing the winner' model. Nevertheless, despite clear effects on prokaryotic abundance, activity and diversity, the diesel degradation was not impacted by top-down control. The present study investigates for the first time the functioning of a complex microbial network (including VLP) using a nutrient-based biostimulation strategy and highlights some key processes useful for tailoring bioremediation.

Self-focusing therapeutic gene delivery with intelligent gene vector swarms: intra-swarm signalling through receptor transgene expression in targeted cells.

Science.gov (United States)

Tolmachov, Oleg E

2015-01-01

Gene delivery in vivo that is tightly focused on the intended target cells is essential to maximize the benefits of gene therapy and to reduce unwanted side-effects. Cell surface markers are immediately available for probing by therapeutic gene vectors and are often used to direct gene transfer with these vectors to specific target cell populations. However, it is not unusual for the choice of available extra-cellular markers to be too scarce to provide a reliable definition of the desired therapeutically relevant set of target cells. Therefore, interrogation of intra-cellular determinants of cell-specificity, such as tissue-specific transcription factors, can be vital in order to provide detailed cell-guiding information to gene vector particles. An important improvement in cell-specific gene delivery can be achieved through auto-buildup in vector homing efficiency using intelligent 'self-focusing' of swarms of vector particles on target cells. Vector self-focusing was previously suggested to rely on the release of diffusible chemo-attractants after a successful target-specific hit by 'scout' vector particles. I hypothesize that intelligent self-focusing behaviour of swarms of cell-targeted therapeutic gene vectors can be accomplished without the employment of difficult-to-use diffusible chemo-attractants, instead relying on the intra-swarm signalling through cells expressing a non-diffusible extra-cellular receptor for the gene vectors. In the proposed model, cell-guiding information is gathered by the 'scout' gene vector particles, which: (1) attach to a variety of cells via a weakly binding (low affinity) receptor; (2) successfully facilitate gene transfer into these cells; (3) query intra-cellular determinants of cell-specificity with their transgene expression control elements and (4) direct the cell-specific biosynthesis of a vector-encoded strongly binding (high affinity) cell-surface receptor. Free members of the vector swarm loaded with therapeutic cargo

Global diversity and biogeography of deep-sea pelagic prokaryotes

KAUST Repository

Salazar, Guillem

2015-08-07

The deep-sea is the largest biome of the biosphere, and contains more than half of the whole ocean\\'s microbes. Uncovering their general patterns of diversity and community structure at a global scale remains a great challenge, as only fragmentary information of deep-sea microbial diversity exists based on regional-scale studies. Here we report the first globally comprehensive survey of the prokaryotic communities inhabiting the bathypelagic ocean using high-throughput sequencing of the 16S rRNA gene. This work identifies the dominant prokaryotes in the pelagic deep ocean and reveals that 50% of the operational taxonomic units (OTUs) belong to previously unknown prokaryotic taxa, most of which are rare and appear in just a few samples. We show that whereas the local richness of communities is comparable to that observed in previous regional studies, the global pool of prokaryotic taxa detected is modest (∼3600 OTUs), as a high proportion of OTUs are shared among samples. The water masses appear to act as clear drivers of the geographical distribution of both particle-attached and free-living prokaryotes. In addition, we show that the deep-oceanic basins in which the bathypelagic realm is divided contain different particle-attached (but not free-living) microbial communities. The combination of the aging of the water masses and a lack of complete dispersal are identified as the main drivers for this biogeographical pattern. All together, we identify the potential of the deep ocean as a reservoir of still unknown biological diversity with a higher degree of spatial complexity than hitherto considered.

Global diversity and biogeography of deep-sea pelagic prokaryotes

KAUST Repository

Salazar, Guillem; Cornejo-Castillo, Francisco M.; Bení tez-Barrios, Veró nica; Fraile-Nuez, Eugenio; Á lvarez-Salgado, X. Antó n; Duarte, Carlos M.; Gasol, Josep M.; Acinas, Silvia G.

2015-01-01

The deep-sea is the largest biome of the biosphere, and contains more than half of the whole ocean's microbes. Uncovering their general patterns of diversity and community structure at a global scale remains a great challenge, as only fragmentary information of deep-sea microbial diversity exists based on regional-scale studies. Here we report the first globally comprehensive survey of the prokaryotic communities inhabiting the bathypelagic ocean using high-throughput sequencing of the 16S rRNA gene. This work identifies the dominant prokaryotes in the pelagic deep ocean and reveals that 50% of the operational taxonomic units (OTUs) belong to previously unknown prokaryotic taxa, most of which are rare and appear in just a few samples. We show that whereas the local richness of communities is comparable to that observed in previous regional studies, the global pool of prokaryotic taxa detected is modest (∼3600 OTUs), as a high proportion of OTUs are shared among samples. The water masses appear to act as clear drivers of the geographical distribution of both particle-attached and free-living prokaryotes. In addition, we show that the deep-oceanic basins in which the bathypelagic realm is divided contain different particle-attached (but not free-living) microbial communities. The combination of the aging of the water masses and a lack of complete dispersal are identified as the main drivers for this biogeographical pattern. All together, we identify the potential of the deep ocean as a reservoir of still unknown biological diversity with a higher degree of spatial complexity than hitherto considered.

BC047440 antisense eukaryotic expression vectors inhibited HepG2 cell proliferation and suppressed xenograft tumorigenicity

International Nuclear Information System (INIS)

Lu, Zheng; Ping, Liang; JianBo, Zhou; XiaoBing, Huang; Yu, Wen; Zheng, Wang; Jing, Li

2012-01-01

The biological functions of the BC047440 gene highly expressed by hepatocellular carcinoma (HCC) are unknown. The objective of this study was to reconstruct antisense eukaryotic expression vectors of the gene for inhibiting HepG 2 cell proliferation and suppressing their xenograft tumorigenicity. The full-length BC047440 cDNA was cloned from human primary HCC by RT-PCR. BC047440 gene fragments were ligated with pMD18-T simple vectors and subsequent pcDNA3.1(+) plasmids to construct the recombinant antisense eukaryotic vector pcDNA3.1(+)BC047440AS. The endogenous BC047440 mRNA abundance in target gene-transfected, vector-transfected and naive HepG 2 cells was semiquantitatively analyzed by RT-PCR and cell proliferation was measured by the MTT assay. Cell cycle distribution and apoptosis were profiled by flow cytometry. The in vivo xenograft experiment was performed on nude mice to examine the effects of antisense vector on tumorigenicity. BC047440 cDNA fragments were reversely inserted into pcDNA3.1(+) plasmids. The antisense vector significantly reduced the endogenous BC047440 mRNA abundance by 41% in HepG 2 cells and inhibited their proliferation in vitro (P < 0.01). More cells were arrested by the antisense vector at the G 1 phase in an apoptosis-independent manner (P = 0.014). Additionally, transfection with pcDNA3.1(+) BC047440AS significantly reduced the xenograft tumorigenicity in nude mice. As a novel cell cycle regulator associated with HCC, the BC047440 gene was involved in cell proliferation in vitro and xenograft tumorigenicity in vivo through apoptosis-independent mechanisms

Differential impact of lytic viruses on prokaryotic morphopopulations in a tropical estuarine system (Cochin estuary, India).

Science.gov (United States)

Jasna, Vijayan; Pradeep Ram, Angia Sriram; Parvathi, Ammini; Sime-Ngando, Telesphore

2018-01-01

Our understanding on the importance of viral lysis in the functioning of tropical estuarine ecosystem is limited. This study examines viral infection of prokaryotes and subsequent lysis of cells belonging to different morphotypes across a salinity gradient in monsoon driven estuarine ecosystem (Cochin estuary, India). High standing stock of viruses and prokaryotes accompanied by lytic infection rates in the euryhaline/mesohaline region of the estuary suggests salinity to have an influential role in driving interactions between prokaryotes and viruses. High prokaryotic mortality rates, up to 42% of prokaryote population in the pre-monsoon season is further substantiated by a high virus to prokaryote ratio (VPR), suggesting that maintenance of a high number of viruses is dependent on the most active fraction of bacterioplankton. Although myoviruses were the dominant viral morphotype (mean = 43%) throughout the study period, there was significant variation among prokaryotic morphotypes susceptible to viral infection. Among them, the viral infected short rod prokaryote morphotype with lower burst estimates (mean = 18 viruses prokaryote-1) was dominant (35%) in the dry seasons whereas a substantial increase in cocci forms (30%) infected by viruses with high burst size (mean = 31 viruses prokaryote-1) was evident during the monsoon season. Such preferential infections of prokaryotic morphopopulations with respect to seasons can have a strong and variable impact on the carbon and energy flow in this tropical ecosystem.

Gateway-compatible vectors for high-throughput protein expression in pro- and eukaryotic cell-free systems.

Science.gov (United States)

Gagoski, Dejan; Mureev, Sergey; Giles, Nichole; Johnston, Wayne; Dahmer-Heath, Mareike; Škalamera, Dubravka; Gonda, Thomas J; Alexandrov, Kirill

2015-02-10

Although numerous techniques for protein expression and production are available the pace of genome sequencing outstrips our ability to analyze the encoded proteins. To address this bottleneck, we have established a system for parallelized cloning, DNA production and cell-free expression of large numbers of proteins. This system is based on a suite of pCellFree Gateway destination vectors that utilize a Species Independent Translation Initiation Sequence (SITS) that mediates recombinant protein expression in any in vitro translation system. These vectors introduce C or N terminal EGFP and mCherry fluorescent and affinity tags, enabling direct analysis and purification of the expressed proteins. To maximize throughput and minimize the cost of protein production we combined Gateway cloning with Rolling Circle DNA Amplification. We demonstrate that as little as 0.1 ng of plasmid DNA is sufficient for template amplification and production of recombinant human protein in Leishmania tarentolae and Escherichia coli cell-free expression systems. Our experiments indicate that this approach can be applied to large gene libraries as it can be reliably performed in multi-well plates. The resulting protein expression pipeline provides a valuable new tool for applications of the post genomic era. Copyright © 2014 Elsevier B.V. All rights reserved.

Expression of variable viruses as herpes simplex glycoprotein D and varicella zoster gE glycoprotein using a novel plasmid based expression system in insect cell

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A.M. Al-Sulaiman

2017-11-01

Full Text Available Several prokaryotic and eukaryotic expression systems have been used for in vitro production of viruses’ proteins. However eukaryotic expression system was always the first choice for production of proteins that undergo post-translational modification such as glycosylation. Recombinant baculoviruses have been widely used as safe vectors to express heterologous genes in the culture of insect cells, but the manipulation involved in creating, titrating, and amplifying viral stocks make it time consuming and laborious. Therefore, to facilitate rapid expression in insect cell, a plasmid based expression system was used to express herpes simplex type 1 glycoprotein D (HSV-1 gD and varicella zoster glycoprotein E (VZV gE. Recombinant plasmids were generated, transfected into insect cells (SF9, and both glycoproteins were expressed 48 h post-infection. A protein with approximately molecular weight of 64-kDa and 98-kDa for HSV-1 gD and VZV gE respectively was expressed and confirmed by SDS. Proteins were detected in insect cells cytoplasm and outer membrane by immunofluorescence. The antigenicity and immunoreactivity of each protein were confirmed by immunoblot and ELISA. Results suggest that this system can be an alternative to the traditional baculovirus expression for small scale expression system in insect cells.

The prokaryote-eukaryote dichotomy: meanings and mythology.

Science.gov (United States)

Sapp, Jan

2005-06-01

Drawing on documents both published and archival, this paper explains how the prokaryote-eukaryote dichotomy of the 1960s was constructed, the purposes it served, and what it implied in terms of classification and phylogeny. In doing so, I first show how the concept was attributed to Edouard Chatton and the context in which he introduced the terms. Following, I examine the context in which the terms were reintroduced into biology in 1962 by Roger Stanier and C. B. van Niel. I study the discourse over the subsequent decade to understand how the organizational dichotomy took on the form of a natural classification as the kingdom Monera or superkingdom Procaryotae. Stanier and van Niel admitted that, in regard to constructing a natural classification of bacteria, structural characteristics were no more useful than physiological properties. They repeatedly denied that bacterial phylogenetics was possible. I thus examine the great historical irony that the "prokaryote," in both its organizational and phylogenetic senses, was defined (negatively) on the basis of structure. Finally, we see how phylogenetic research based on 16S rRNA led by Carl Woese and his collaborators confronted the prokaryote concept while moving microbiology to the center of evolutionary biology.

Effects of viruses and predators on prokaryotic community composition.

Science.gov (United States)

Jardillier, Ludwig; Bettarel, Yvan; Richardot, Mathilde; Bardot, Corinne; Amblard, Christian; Sime-Ngando, Télesphore; Debroas, Didier

2005-11-01

Dialysis bags were used to examine the impact of predation and viral lysis on prokaryotic community composition (PCC) over a 5-day experiment in the oligomesotrophic Lake Pavin (France). The impact of the different predator communities (protists and metazoans) of prokaryotes was estimated by water fractionation (protists, which also affected PCC, whereas viruses seemed to be essentially responsible for profound changes in PCC via direct and indirect actions.

Prokaryote metabolism activity

OpenAIRE

Biederman, Lori

2017-01-01

I wrote this activity to emphasize that prokaryotic organisms can carry out 6 different types of metabolisms (as presented in Freeman’s Biological Science textbook) and this contrasts to eukaryotes, which can only use 2 metabolism pathways (photoautotroph and heterotroph).    For in class materials I remove the  red box (upper right corner) and print slides 3-10, place them back-to-back and laminate them.  The students get a key (slide 2) and a two-sided organism sheet...

Production of isotopically labeled heterologous proteins in non-E. coli prokaryotic and eukaryotic cells

International Nuclear Information System (INIS)

Takahashi, Hideo; Shimada, Ichio

2010-01-01

The preparation of stable isotope-labeled proteins is necessary for the application of a wide variety of NMR methods, to study the structures and dynamics of proteins and protein complexes. The E. coli expression system is generally used for the production of isotope-labeled proteins, because of the advantages of ease of handling, rapid growth, high-level protein production, and low cost for isotope-labeling. However, many eukaryotic proteins are not functionally expressed in E. coli, due to problems related to disulfide bond formation, post-translational modifications, and folding. In such cases, other expression systems are required for producing proteins for biomolecular NMR analyses. In this paper, we review the recent advances in expression systems for isotopically labeled heterologous proteins, utilizing non-E. coli prokaryotic and eukaryotic cells.

A novel artificial microRNA expressing AAV vector for phospholamban silencing in cardiomyocytes improves Ca2+ uptake into the sarcoplasmic reticulum.

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Tobias Gröβl

Full Text Available In failing rat hearts, post-transcriptonal inhibition of phospholamban (PLB expression by AAV9 vector-mediated cardiac delivery of short hairpin RNAs directed against PLB (shPLBr improves both impaired SERCA2a controlled Ca2+ cycling and contractile dysfunction. Cardiac delivery of shPLB, however, was reported to cause cardiac toxicity in canines. Thus we developed a new AAV vector, scAAV6-amiR155-PLBr, expressing a novel engineered artificial microRNA (amiR155-PLBr directed against PLB under control of a heart-specific hybrid promoter. Its PLB silencing efficiency and safety were compared with those of an AAV vector expressing shPLBr (scAAV6-shPLBr from an ubiquitously active U6 promoter. Investigations were carried out in cultured neonatal rat cardiomyocytes (CM over a period of 14 days. Compared to shPLBr, amiR155-PLBr was expressed at a significantly lower level, resulting in delayed and less pronounced PLB silencing. Despite decreased knockdown efficiency of scAAV6-amiR155-PLBr, a similar increase of the SERCA2a-catalyzed Ca2+ uptake into sarcoplasmic reticulum (SR vesicles was observed for both the shPLBr and amiR155-PLBr vectors. Proteomic analysis confirmed PLB silencing of both therapeutic vectors and revealed that shPLBr, but not the amiR155-PLBr vector, increased the proinflammatory proteins STAT3, STAT1 and activated STAT1 phosphorylation at the key amino acid residue Tyr701. Quantitative RT-PCR analysis detected alterations in the expression of several cardiac microRNAs after treatment of CM with scAAV6-shPLBr and scAAV6-amiR155-PLBr, as well as after treatment with its related amiR155- and shRNAs-expressing control AAV vectors. The results demonstrate that scAAV6-amiR155-PLBr is capable of enhancing the Ca2+ transport function of the cardiac SR PLB/SERCA2a system as efficiently as scAAV6-shPLBr while offering a superior safety profile.

Improved innate and adaptive immunostimulation by genetically modified HIV-1 protein expressing NYVAC vectors.

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Esther D Quakkelaar

Full Text Available Attenuated poxviruses are safe and capable of expressing foreign antigens. Poxviruses are applied in veterinary vaccination and explored as candidate vaccines for humans. However, poxviruses express multiple genes encoding proteins that interfere with components of the innate and adaptive immune response. This manuscript describes two strategies aimed to improve the immunogenicity of the highly attenuated, host-range restricted poxvirus NYVAC: deletion of the viral gene encoding type-I interferon-binding protein and development of attenuated replication-competent NYVAC. We evaluated these newly generated NYVAC mutants, encoding HIV-1 env, gag, pol and nef, for their ability to stimulate HIV-specific CD8 T-cell responses in vitro from blood mononuclear cells of HIV-infected subjects. The new vectors were evaluated and compared to the parental NYVAC vector in dendritic cells (DCs, RNA expression arrays, HIV gag expression and cross-presentation assays in vitro. Deletion of type-I interferon-binding protein enhanced expression of interferon and interferon-induced genes in DCs, and increased maturation of infected DCs. Restoration of replication competence induced activation of pathways involving antigen processing and presentation. Also, replication-competent NYVAC showed increased Gag expression in infected cells, permitting enhanced cross-presentation to HIV-specific CD8 T cells and proliferation of HIV-specific memory CD8 T-cells in vitro. The recombinant NYVAC combining both modifications induced interferon-induced genes and genes involved in antigen processing and presentation, as well as increased Gag expression. This combined replication-competent NYVAC is a promising candidate for the next generation of HIV vaccines.

Prokaryotic caspase homologs: phylogenetic patterns and functional characteristics reveal considerable diversity.

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Johannes Asplund-Samuelsson

Full Text Available Caspases accomplish initiation and execution of apoptosis, a programmed cell death process specific to metazoans. The existence of prokaryotic caspase homologs, termed metacaspases, has been known for slightly more than a decade. Despite their potential connection to the evolution of programmed cell death in eukaryotes, the phylogenetic distribution and functions of these prokaryotic metacaspase sequences are largely uncharted, while a few experiments imply involvement in programmed cell death. Aiming at providing a more detailed picture of prokaryotic caspase homologs, we applied a computational approach based on Hidden Markov Model search profiles to identify and functionally characterize putative metacaspases in bacterial and archaeal genomes. Out of the total of 1463 analyzed genomes, merely 267 (18% were identified to contain putative metacaspases, but their taxonomic distribution included most prokaryotic phyla and a few archaea (Euryarchaeota. Metacaspases were particularly abundant in Alphaproteobacteria, Deltaproteobacteria and Cyanobacteria, which harbor many morphologically and developmentally complex organisms, and a distinct correlation was found between abundance and phenotypic complexity in Cyanobacteria. Notably, Bacillus subtilis and Escherichia coli, known to undergo genetically regulated autolysis, lacked metacaspases. Pfam domain architecture analysis combined with operon identification revealed rich and varied configurations among the metacaspase sequences. These imply roles in programmed cell death, but also e.g. in signaling, various enzymatic activities and protein modification. Together our data show a wide and scattered distribution of caspase homologs in prokaryotes with structurally and functionally diverse sub-groups, and with a potentially intriguing evolutionary role. These features will help delineate future characterizations of death pathways in prokaryotes.

Recombinant human parvovirus B19 vectors: erythroid cell-specific delivery and expression of transduced genes.

Science.gov (United States)

Ponnazhagan, S; Weigel, K A; Raikwar, S P; Mukherjee, P; Yoder, M C; Srivastava, A

1998-06-01

A novel packaging strategy combining the salient features of two human parvoviruses, namely the pathogenic parvovirus B19 and the nonpathogenic adeno-associated virus type 2 (AAV), was developed to achieve erythroid cell-specific delivery as well as expression of the transduced gene. The development of such a chimeric vector system was accomplished by packaging heterologous DNA sequences cloned within the inverted terminal repeats of AAV and subsequently packaging the DNA inside the capsid structure of B19 virus. Recombinant B19 virus particles were assembled, as evidenced by electron microscopy as well as DNA slot blot analyses. The hybrid vector failed to transduce nonerythroid human cells, such as 293 cells, as expected. However, MB-02 cells, a human megakaryocytic leukemia cell line which can be infected by B19 virus following erythroid differentiation with erythropoietin (N. C. Munshi, S. Z. Zhou, M. J. Woody, D. A. Morgan, and A. Srivastava, J. Virol. 67:562-566, 1993) but lacks the putative receptor for AAV (S. Ponnazhagan, X.-S. Wang, M. J. Woody, F. Luo, L. Y. Kang, M. L. Nallari, N. C. Munshi, S. Z. Zhou, and A. Srivastava, J. Gen. Virol. 77:1111-1122, 1996), were readily transduced by this vector. The hybrid vector was also found to specifically target the erythroid population in primary human bone marrow cells as well as more immature hematopoietic progenitor cells following erythroid differentiation, as evidenced by selective expression of the transduced gene in these target cells. Preincubation with anticapsid antibodies against B19 virus, but not anticapsid antibodies against AAV, inhibited transduction of primary human erythroid cells. The efficiency of transduction of primary human erythroid cells by the recombinant B19 virus vector was significantly higher than that by the recombinant AAV vector. Further development of the AAV-B19 virus hybrid vector system should prove beneficial in gene therapy protocols aimed at the correction of inherited and

Recombinant Human Parvovirus B19 Vectors: Erythroid Cell-Specific Delivery and Expression of Transduced Genes

Science.gov (United States)

Ponnazhagan, Selvarangan; Weigel, Kirsten A.; Raikwar, Sudhanshu P.; Mukherjee, Pinku; Yoder, Mervin C.; Srivastava, Arun

1998-01-01

A novel packaging strategy combining the salient features of two human parvoviruses, namely the pathogenic parvovirus B19 and the nonpathogenic adeno-associated virus type 2 (AAV), was developed to achieve erythroid cell-specific delivery as well as expression of the transduced gene. The development of such a chimeric vector system was accomplished by packaging heterologous DNA sequences cloned within the inverted terminal repeats of AAV and subsequently packaging the DNA inside the capsid structure of B19 virus. Recombinant B19 virus particles were assembled, as evidenced by electron microscopy as well as DNA slot blot analyses. The hybrid vector failed to transduce nonerythroid human cells, such as 293 cells, as expected. However, MB-02 cells, a human megakaryocytic leukemia cell line which can be infected by B19 virus following erythroid differentiation with erythropoietin (N. C. Munshi, S. Z. Zhou, M. J. Woody, D. A. Morgan, and A. Srivastava, J. Virol. 67:562–566, 1993) but lacks the putative receptor for AAV (S. Ponnazhagan, X.-S. Wang, M. J. Woody, F. Luo, L. Y. Kang, M. L. Nallari, N. C. Munshi, S. Z. Zhou, and A. Srivastava, J. Gen. Virol. 77:1111–1122, 1996), were readily transduced by this vector. The hybrid vector was also found to specifically target the erythroid population in primary human bone marrow cells as well as more immature hematopoietic progenitor cells following erythroid differentiation, as evidenced by selective expression of the transduced gene in these target cells. Preincubation with anticapsid antibodies against B19 virus, but not anticapsid antibodies against AAV, inhibited transduction of primary human erythroid cells. The efficiency of transduction of primary human erythroid cells by the recombinant B19 virus vector was significantly higher than that by the recombinant AAV vector. Further development of the AAV-B19 virus hybrid vector system should prove beneficial in gene therapy protocols aimed at the correction of inherited

[Construction and expression of recombinant lentiviral vectors of AKT2,PDK1 and BAD].

Science.gov (United States)

Zhu, Jing; Chen, Bo-Jiang; Huang, Na; Li, Wei-Min

2014-03-01

To construct human protein kinase B (ATK2), phosphoinositide-dependent kinase 1 (PDK1) and bcl-2-associated death protein (BAD) lentiviral expression vector, and to determine their expressions in 293T cells. Total RNA was extracted from lung cancer tissues. The full-length coding regions of human ATK2, BAD and PDK1 cDNA were amplified via RT-PCR using specific primers, subcloned into PGEM-Teasy and then sequenced for confirmation. The full-length coding sequence was cut out with a specific restriction enzyme digest and subclone into pCDF1-MCS2-EF1-copGFP. The plasmids were transfected into 293T cells using the calcium phosphate method. The over expression of AKT2, BAD and PDK1 were detected by Western blot. AKT2, PDK1 and BAD were subcloned into pCDF1-MCS2-EF1-copGFP, with an efficiency of transfection of 100%, 95%, and 90% respectively. The virus titers were 6.7 x 10(6) PFU/mL in the supernatant. After infection, the proteins of AKT2, PDK1 and BAD were detected by Western blot. The lentivial vector pCDF1-MCS2-EF1-copGFP containing AKT2, BAD and PDK1 were successfully constructed and expressed in 293T cells.

Viral infections stimulate the metabolism and shape prokaryotic assemblages in submarine mud volcanoes.

Science.gov (United States)

Corinaldesi, Cinzia; Dell'Anno, Antonio; Danovaro, Roberto

2012-06-01

Mud volcanoes are geological structures in the oceans that have key roles in the functioning of the global ecosystem. Information on the dynamics of benthic viruses and their interactions with prokaryotes in mud volcano ecosystems is still completely lacking. We investigated the impact of viral infection on the mortality and assemblage structure of benthic prokaryotes of five mud volcanoes in the Mediterranean Sea. Mud volcano sediments promote high rates of viral production (1.65-7.89 × 10(9) viruses g(-1) d(-1)), viral-induced prokaryotic mortality (VIPM) (33% cells killed per day) and heterotrophic prokaryotic production (3.0-8.3 μgC g(-1) d(-1)) when compared with sediments outside the mud volcano area. The viral shunt (that is, the microbial biomass converted into dissolved organic matter as a result of viral infection, and thus diverted away from higher trophic levels) provides 49 mgC m(-2) d(-1), thus fuelling the metabolism of uninfected prokaryotes and contributing to the total C budget. Bacteria are the dominant components of prokaryotic assemblages in surface sediments of mud volcanoes, whereas archaea dominate the subsurface sediment layers. Multivariate multiple regression analyses show that prokaryotic assemblage composition is not only dependant on the geochemical features and processes of mud volcano ecosystems but also on synergistic interactions between bottom-up (that is, trophic resources) and top-down (that is, VIPM) controlling factors. Overall, these findings highlight the significant role of the viral shunt in sustaining the metabolism of prokaryotes and shaping their assemblage structure in mud volcano sediments, and they provide new clues for our understanding of the functioning of cold-seep ecosystems.

TimesVector: a vectorized clustering approach to the analysis of time series transcriptome data from multiple phenotypes.

Science.gov (United States)

Jung, Inuk; Jo, Kyuri; Kang, Hyejin; Ahn, Hongryul; Yu, Youngjae; Kim, Sun

2017-12-01

Identifying biologically meaningful gene expression patterns from time series gene expression data is important to understand the underlying biological mechanisms. To identify significantly perturbed gene sets between different phenotypes, analysis of time series transcriptome data requires consideration of time and sample dimensions. Thus, the analysis of such time series data seeks to search gene sets that exhibit similar or different expression patterns between two or more sample conditions, constituting the three-dimensional data, i.e. gene-time-condition. Computational complexity for analyzing such data is very high, compared to the already difficult NP-hard two dimensional biclustering algorithms. Because of this challenge, traditional time series clustering algorithms are designed to capture co-expressed genes with similar expression pattern in two sample conditions. We present a triclustering algorithm, TimesVector, specifically designed for clustering three-dimensional time series data to capture distinctively similar or different gene expression patterns between two or more sample conditions. TimesVector identifies clusters with distinctive expression patterns in three steps: (i) dimension reduction and clustering of time-condition concatenated vectors, (ii) post-processing clusters for detecting similar and distinct expression patterns and (iii) rescuing genes from unclassified clusters. Using four sets of time series gene expression data, generated by both microarray and high throughput sequencing platforms, we demonstrated that TimesVector successfully detected biologically meaningful clusters of high quality. TimesVector improved the clustering quality compared to existing triclustering tools and only TimesVector detected clusters with differential expression patterns across conditions successfully. The TimesVector software is available at http://biohealth.snu.ac.kr/software/TimesVector/. sunkim.bioinfo@snu.ac.kr. Supplementary data are available at

Cloning and Expression of ORFV B2L Gene%羊口疮病毒B2L基因克隆及表达

Institute of Scientific and Technical Information of China (English)

赵文博; 李瑞航; 贺鹏亮; 李朋娅; 王梦醒; 杨春华; 于永忠; 崔玉东

2015-01-01

为了获得羊口疮病毒囊膜蛋白。以羊口疮病毒基因组为模板，利用羊口疮病毒B2L基因特异性引物，进行PCR扩增，回收以及纯化PCR产物，连接到PMD18-T载体上，测序结果表明B2L基因全长1137 bp，编码379个氨基酸。再将B2L基因克隆至pET-30a，构建原核表达载体pET30a-B2L。经菌液PCR、酶切鉴定以及SDS-PAGE等方法表明成功构建原核表达载体，这将为今后的单抗制备以及羊口疮病毒的疫苗研究打下基础。%To obtain ORFV envelope protein and take ORFV genome as template,ORFV B2L gene was used as specific primers to amplify PCR and recovery and purify PCR products. Connected PCR products to PMD18-T vector,the sequence result showed that full-length of B2L gene was 1137 bp,and Encoding was 379 amino acids. B2L gene was cloned into pET-30a to construct prokaryotic expression vector pET30a-B2L. Prokaryotic expression vector was constructed by the methods of bacterium liquid PCR, enzyme identification and SDS-PAGE,which would provide the research basis of monoclonal antibody and ORFV vaccine in the future.

Novel redox nanomedicine improves gene expression of polyion complex vector

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Kazuko Toh, Toru Yoshitomi, Yutaka Ikeda and Yukio Nagasaki

2011-01-01

Full Text Available Gene therapy has generated worldwide attention as a new medical technology. While non-viral gene vectors are promising candidates as gene carriers, they have several issues such as toxicity and low transfection efficiency. We have hypothesized that the generation of reactive oxygen species (ROS affects gene expression in polyplex supported gene delivery systems. The effect of ROS on the gene expression of polyplex was evaluated using a nitroxide radical-containing nanoparticle (RNP as an ROS scavenger. When polyethyleneimine (PEI/pGL3 or PEI alone was added to the HeLa cells, ROS levels increased significantly. In contrast, when (PEI/pGL3 or PEI was added with RNP, the ROS levels were suppressed. The luciferase expression was increased by the treatment with RNP in a dose-dependent manner and the cellular uptake of pDNA was also increased. Inflammatory cytokines play an important role in ROS generation in vivo. In particular, tumor necrosis factor (TNF-α caused intracellular ROS generation in HeLa cells and decreased gene expression. RNP treatment suppressed ROS production even in the presence of TNF-α and increased gene expression. This anti-inflammatory property of RNP suggests that it may be used as an effective adjuvant for non-viral gene delivery systems.

A molecular toolbox for rapid generation of viral vectors to up- or down-regulate in vivo neuronal gene expression

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Melanie D. White

2011-07-01

Full Text Available We introduce a molecular toolbox for manipulation of neuronal gene expression in vivo. The toolbox includes promoters, ion channels, optogenetic tools, fluorescent proteins and intronic artificial microRNAs. The components are easily assembled into adeno-associated virus (AAV or lentivirus vectors using recombination cloning. We demonstrate assembly of toolbox components into lentivirus and AAV vectors and use these vectors for in vivo expression of inwardly rectifying potassium channels (Kir2.1, Kir3.1 and Kir3.2 and an artificial microRNA targeted against the ion channel HCN1 (HCN1 miR. We show that AAV assembled to express HCN1 miR produces efficacious and specific in vivo knockdown of HCN1 channels. Comparison of in vivo viral transduction using HCN1 miR with mice containing a germ line deletion of HCN1 reveals similar physiological phenotypes in cerebellar Purkinje cells. The easy assembly and re-usability of the toolbox components, together with the ability to up- or down-regulate neuronal gene expression in vivo, may be useful for applications in many areas of neuroscience.

Molecular cloning and characterization of a cytoplasmic cyclophilin ...

African Journals Online (AJOL)

Jane

2011-08-08

Aug 8, 2011 ... In real-time qPCR analysis, the Sc-CyP gene showed induced expression under ... signaling pathway was also existed in plants (Luan et al.,. 1993). ... Construction of prokaryotic expression vector of Sc-CyP gene. pET29a (+) ...

Prokaryotic Expression, Purification and Characterization of a Novel Rice Seed Lipoxygenase Gene OsLOX1

Directory of Open Access Journals (Sweden)

Ren Wang

2008-06-01

Full Text Available Lipoxygenase (LOX, EC1.13.11.12 is a key enzyme during the degradation of lipids in animals and even plants, and also the first key enzyme responsible for the biosynthesis of jasmonate. To purify and characterize the OsLOX1 gene from rice seeds, the entire coding region of the OsLOX1 gene was inserted into an expression vector pET30a(+ and transformed into Escherichia coli BL21 (DE3. Expression of the fusion protein was successfully induced by isopropyl-β-D- thiogalactopyranoside (IPTG and the purified recombinant protein was obtained by His·Bind® Kits. Further assay showed that the purified recombinant protein exhibited the LOX activity. The optimum pH was 4.8 (acetate buffer and the optimum temperature was 30°C for the above enzyme. Thus, the recombinant might confer an available usage for the synthesis of jasmonate in vitro, and also provides a possibility for elucidating the inter-relationship between the primary structure of the plant seed lipoxygenase protein and its physiological functions.

Prokaryotes versus Eukaryotes: Who is hosting whom?

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Guillermo eTellez

2014-10-01

Full Text Available Microorganisms represent the largest component of biodiversity in our world. For millions of years, prokaryotic microorganisms have functioned as a major selective force shaping eukaryotic evolution. Microbes that live inside and on animals outnumber the animals’ actual somatic and germ cells by an estimated 10-fold. Collectively, the intestinal microbiome represents a ‘forgotten organ’, functioning as an organ inside another that can execute many physiological responsibilities. The nature of primitive eukaryotes was drastically changed due to the association with symbiotic prokaryotes facilitating mutual coevolution of host and microbe. Phytophagous insects have long been used to test theories of evolutionary diversification; moreover, the diversification of a number of phytophagous insect lineages has been linked to mutualisms with microbes. From termites and honey bees to ruminants and mammals, depending on novel biochemistries provided by the prokaryotic microbiome, the association helps to metabolize several nutrients that the host cannot digest and converting these into useful end products (such as short chain fatty acids, a process which has huge impact on the biology and homeostasis of metazoans. More importantly, in a direct and/or indirect way, the intestinal microbiota influences the assembly of gut-associated lymphoid tissue, helps to educate immune system, affects the integrity of the intestinal mucosal barrier, modulates proliferation and differentiation of its epithelial lineages, regulates angiogenesis, and modifies the activity of enteric as well as the central nervous system,. Despite these important effects, the mechanisms by which the gut microbial community influences the host’s biology remains almost entirely unknown. Our aim here is to encourage empirical inquiry into the relationship between mutualism and evolutionary diversification between prokaryotes and eukaryotes which encourage us to postulate: Who is

Biodegradation of Emiliania huxleyi Aggregates by natural Prokaryotic Communities under Increasing Hydrostatic Pressure.

Science.gov (United States)

Riou, V.; Para, J.; Garel, M.; Guigue, C.; Al Ali, B.; Santinelli, C.; Lefèvre, D.; Gattuso, J. P.; Goutx, M.; Panagiotopoulos, C.; Beaufort, L.; Jacquet, S.; Le Moigne, F. A. C.; Tachikawa, K.; Tamburini, C.

2016-02-01

Fluxes of particulate organic carbon (POC) and minerals are positively correlated, suggesting that minerals could enhance the flux of POC into the deep ocean. The so called "ballast effect" posits that minerals could increase sinking particle densities and/or protect the organic matter from heterotrophic degradation. Laboratory controlled experiments on coccolithophorid aggregates under atmospheric pressure show that biogenic calcite both increases particle settling velocities and preserves the organic matter. However, such experiments have yet to include genuine prokaryote rates indicators as well as the effect of increasing pressure. Here, we used the PArticle Sinking Simulator (PASS) to investigate the effect of the increasing pressure on the degradation of Emiliania huxleyi (calcifiers) aggregates. Extra care was taken to obtain culture aggregates with low prokaryotic abundance prior to exposure to natural mesopelagic prokaryotic communities. Particulate organic and inorganic carbon and dissolved organic carbon concentrations were monitored along with the lipid and carbohydrate compositions, as well as prokaryotic community abundance and specific diversity. A control experiment, without natural prokaryotic community addition, indicates that the pressure increase did not have any effect on calcite dissolution observed after ten days. In contrast, the addition of natural prokaryotic community accelerates calcite dissolution under conditions of increasing pressure. Prokaryotic community development and the lipid fraction of E. huxleyi particulate organic carbon are enhanced under increasing pressure. These results suggest that hydrostatic pressure denatures the structural integrity of the carbonate skeleton that protects the cellular organic matter.

Gateway-assisted vector construction to facilitate expression of foreign proteins in the chloroplast of single celled algae.

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Melanie Oey

Full Text Available With a rising world population, demand will increase for food, energy and high value products. Renewable production systems, including photosynthetic microalgal biotechnologies, can produce biomass for foods, fuels and chemical feedstocks and in parallel allow the production of high value protein products, including recombinant proteins. Such high value recombinant proteins offer important economic benefits during startup of industrial scale algal biomass and biofuel production systems, but the limited markets for individual recombinant proteins will require a high throughput pipeline for cloning and expression in microalgae, which is currently lacking, since genetic engineering of microalgae is currently complex and laborious. We have introduced the recombination based Gateway® system into the construction process of chloroplast transformation vectors for microalgae. This simplifies the vector construction and allows easy, fast and flexible vector design for the high efficiency protein production in microalgae, a key step in developing such expression pipelines.

Protection against California 2002 NDV strain afforded by adenovirus vectored vaccine expressing Fusion or Hemagglutination-neuraminidase genes

Science.gov (United States)

Vectored vaccines expressing the combination of the hemagglutinin-neuraminidase (HN) and fusion (F) genes generally have better clinical protection against Newcastle disease virus (NDV) than when either the F and HN genes are expressed alone. Interestingly, the protection induced by F is usually bet...

Engineering prokaryotic transcriptional activators as metabolite biosensors in yeast

DEFF Research Database (Denmark)

Skjødt, Mette Louise; Snoek, Tim; Kildegaard, Kanchana Rueksomtawin

2016-01-01

,cis-muconic acid at different levels, and found that reporter gene output correlated with production. The transplantation of prokaryotic transcriptional activators into the eukaryotic chassis illustrates the potential of a hitherto untapped biosensor resource useful for biotechnological applications....... real-time monitoring of production has attracted attention. Here we applied systematic engineering of multiple parameters to search for a general biosensor design in the budding yeast Saccharomyces cerevisiae based on small-molecule binding transcriptional activators from the prokaryote superfamily...

Construction of a Shuttle Vector for Heterologous Expression of a Novel Fungal α-Amylase Gene in Aspergillus oryzae.

Science.gov (United States)

Yin, Yanchen; Mao, Youzhi; Yin, Xiaolie; Gao, Bei; Wei, Dongzhi

2015-07-01

The filamentous fungus Aspergillus oryzae is a well-known expression host used to express homologous and heterologous proteins in a number of industrial applications. To facilitate higher yields of proteins of interest, we constructed the pAsOP vector to express heterologous proteins in A. oryzae. pAsOP carries a selectable marker, pyrG, derived from Aspergillus nidulans, and a strong promoter and a terminator of the amyB gene derived from A. oryzae. pAsOP transformed A. oryzae efficiently via the PEG-CaCl2-mediated transformation method. As proof of concept, green fluorescent protein (GFP) was successfully expressed in A. oryzae transformed by pAsOP-GFP. Additionally, we identified a novel fungal α-amylase (PcAmy) gene from Penicillium sp. and cloned the gene into the vector. After transformation by pAsOPPcAmy, the α-amylase PcAmy from Penicillium sp. was successfully expressed in a heterologous host system for the first time. The α-amylase activity in the A. oryzae transformant was increased by 62.3% compared with the untransformed A. oryzae control. The PcAmy protein produced in the system had an optimum pH of 5.0 and optimum temperature of 30°C. As a cold-adapted enzyme, PcAmy shows potential value in industrial applications because of its high catalytic activity at low temperature. Furthermore, the expression vector reported in this study provides promising utility for further scientific research and biotechnological applications.

PairWise Neighbours database: overlaps and spacers among prokaryote genomes

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Garcia-Vallvé Santiago

2009-06-01

Full Text Available Abstract Background Although prokaryotes live in a variety of habitats and possess different metabolic and genomic complexity, they have several genomic architectural features in common. The overlapping genes are a common feature of the prokaryote genomes. The overlapping lengths tend to be short because as the overlaps become longer they have more risk of deleterious mutations. The spacers between genes tend to be short too because of the tendency to reduce the non coding DNA among prokaryotes. However they must be long enough to maintain essential regulatory signals such as the Shine-Dalgarno (SD sequence, which is responsible of an efficient translation. Description PairWise Neighbours is an interactive and intuitive database used for retrieving information about the spacers and overlapping genes among bacterial and archaeal genomes. It contains 1,956,294 gene pairs from 678 fully sequenced prokaryote genomes and is freely available at the URL http://genomes.urv.cat/pwneigh. This database provides information about the overlaps and their conservation across species. Furthermore, it allows the wide analysis of the intergenic regions providing useful information such as the location and strength of the SD sequence. Conclusion There are experiments and bioinformatic analysis that rely on correct annotations of the initiation site. Therefore, a database that studies the overlaps and spacers among prokaryotes appears to be desirable. PairWise Neighbours database permits the reliability analysis of the overlapping structures and the study of the SD presence and location among the adjacent genes, which may help to check the annotation of the initiation sites.

Advances in algal-prokaryotic wastewater treatment: A review of nitrogen transformations, reactor configurations and molecular tools.

Science.gov (United States)

Wang, Meng; Keeley, Ryan; Zalivina, Nadezhda; Halfhide, Trina; Scott, Kathleen; Zhang, Qiong; van der Steen, Peter; Ergas, Sarina J

2018-07-01

The synergistic activity of algae and prokaryotic microorganisms can be used to improve the efficiency of biological wastewater treatment, particularly with regards to nitrogen removal. For example, algae can provide oxygen through photosynthesis needed for aerobic degradation of organic carbon and nitrification and harvested algal-prokaryotic biomass can be used to produce high value chemicals or biogas. Algal-prokaryotic consortia have been used to treat wastewater in different types of reactors, including waste stabilization ponds, high rate algal ponds and closed photobioreactors. This review addresses the current literature and identifies research gaps related to the following topics: 1) the complex interactions between algae and prokaryotes in wastewater treatment; 2) advances in bioreactor technologies that can achieve high nitrogen removal efficiencies in small reactor volumes, such as algal-prokaryotic biofilm reactors and enhanced algal-prokaryotic treatment systems (EAPS); 3) molecular tools that have expanded our understanding of the activities of algal and prokaryotic communities in wastewater treatment processes. Copyright © 2018 Elsevier Ltd. All rights reserved.

Plasmid and chromosome segregation in prokaryotes

DEFF Research Database (Denmark)

Møller-Jensen, Jakob; Bugge Jensen, Rasmus; Gerdes, Kenn

2000-01-01

Recent major advances in the understanding of prokaryotic DNA segregation have been achieved by using fluorescence microscopy to visualize the localization of cellular components. Plasmids and bacterial chromosomes are partitioned in a highly dynamic fashion, suggesting the presence of a mitotic...

[Selection and construction of cell line stably expressing survivin gene in lower level through eukaryotic plasmid vector of shRNA].

Science.gov (United States)

Wang, Wen-Xia; Sun, Shan-Zhen; Song, Ying

2008-06-01

To construct a short hairpin RNA(shRNA) interference expression plasmid vector of survivin gene, transfect tongue squamous cell carcinoma line Tca8113 which expressed survivin gene in a high level, and choose the cells whose survivin gene were suppressed significantly. Two pairs of oligonucleotide sequences specific for survivin gene were designed and synthesized, and cloned into pSilencer-2.1U6-neo plasmid. The recombinant plasmids (named PS1 and PS2) were amplified in Ecoli. DH5alpha was identified by restriction digestion, PCR and sequencing. The vectors were transfected into Tca8113 cells with lipofectamine 2000. After selection with G418, the stable cell clones were attained. Survivn expression was assayed with real-time quantitative PCR and Western blotting. SAS8.0 software package was used for Student t test. Two vectors were constructed successfully and stable cell clones with PS1 or PS2 plasmid were obtained. As compared with those of control, survivin expression of transfected cell with PS1 or PS2 in mRNA level was significantly suppressed (P<0.05). In protein level, only those of transfected cell with PS2 was significantly suppressed (P<0.01). The shRNA interference expression plasmid vectors of survivin gene are successfully constructed, and Tca8113 cells which express survivin gene in a stable lower level are attained, which enable us to carry out further research on gene therapy of oral squamous cell carcinoma. Supported by National Natural Science Foundation of China (Grant No.30572056).

A Vector with a Single Promoter for In Vitro Transcription and Mammalian Cell Expression of CRISPR gRNAs.

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Peter J Romanienko

Full Text Available The genomes of more than 50 organisms have now been manipulated due to rapid advancement of gene editing technology. One way to perform gene editing in the mouse using the CRISPR/CAS system, guide RNA (gRNA and CAS9 mRNA transcribed in vitro are microinjected into fertilized eggs that are then allowed to develop to term. As a rule, gRNAs are tested first in tissue culture cells and the one with the highest locus-specific cleavage activity is chosen for microinjection. For cell transfections, gRNAs are typically expressed using the human U6 promoter (hU6. However, gRNAs for microinjection into zygotes are obtained by in vitro transcription from a T7 bacteriophage promoter in a separate plasmid vector. Here, we describe the design and construction of a combined U6T7 hybrid promoter from which the same gRNA sequence can be expressed. An expression vector containing such a hybrid promoter can now be used to generate gRNA for testing in mammalian cells as well as for microinjection purposes. The gRNAs expressed and transcribed from this vector are found to be functional in cells as well as in mice.

Vector modifications to eliminate transposase expression following piggyBac-mediated transgenesis

Science.gov (United States)

Chakraborty, Syandan; Ji, HaYeun; Chen, Jack; Gersbach, Charles A.; Leong, Kam W.

2014-01-01

Transgene insertion plays an important role in gene therapy and in biological studies. Transposon-based systems that integrate transgenes by transposase-catalyzed “cut-and-paste” mechanism have emerged as an attractive system for transgenesis. Hyperactive piggyBac transposon is particularly promising due to its ability to integrate large transgenes with high efficiency. However, prolonged expression of transposase can become a potential source of genotoxic effects due to uncontrolled transposition of the integrated transgene from one chromosomal locus to another. In this study we propose a vector design to decrease post-transposition expression of transposase and to eliminate the cells that have residual transposase expression. We design a single plasmid construct that combines the transposase and the transpositioning transgene element to share a single polyA sequence for termination. Consequently, the separation of the transposase element from the polyA sequence after transposition leads to its deactivation. We also co-express Herpes Simplex Virus thymidine kinase (HSV-tk) with the transposase. Therefore, cells having residual transposase expression can be eliminated by the administration of ganciclovir. We demonstrate the utility of this combination transposon system by integrating and expressing a model therapeutic gene, human coagulation Factor IX, in HEK293T cells. PMID:25492703

Emerging spatial patterns in Antarctic prokaryotes.

Science.gov (United States)

Chong, Chun-Wie; Pearce, David A; Convey, Peter

2015-01-01

Recent advances in knowledge of patterns of biogeography in terrestrial eukaryotic organisms have led to a fundamental paradigm shift in understanding of the controls and history of life on land in Antarctica, and its interactions over the long term with the glaciological and geological processes that have shaped the continent. However, while it has long been recognized that the terrestrial ecosystems of Antarctica are dominated by microbes and their processes, knowledge of microbial diversity and distributions has lagged far behind that of the macroscopic eukaryote organisms. Increasing human contact with and activity in the continent is leading to risks of biological contamination and change in a region whose isolation has protected it for millions of years at least; these risks may be particularly acute for microbial communities which have, as yet, received scant recognition and attention. Even a matter apparently as straightforward as Protected Area designation in Antarctica requires robust biodiversity data which, in most parts of the continent, remain almost completely unavailable. A range of important contributing factors mean that it is now timely to reconsider the state of knowledge of Antarctic terrestrial prokaryotes. Rapid advances in molecular biological approaches are increasingly demonstrating that bacterial diversity in Antarctica may be far greater than previously thought, and that there is overlap in the environmental controls affecting both Antarctic prokaryotic and eukaryotic communities. Bacterial dispersal mechanisms and colonization patterns remain largely unaddressed, although evidence for regional evolutionary differentiation is rapidly accruing and, with this, there is increasing appreciation of patterns in regional bacterial biogeography in this large part of the globe. In this review, we set out to describe the state of knowledge of Antarctic prokaryote diversity patterns, drawing analogy with those of eukaryote groups where appropriate

Genome resource utilization during prokaryotic development

Czech Academy of Sciences Publication Activity Database

Vohradský, Jiří; Ramsden, J. J.

2001-01-01

Roč. 15, - (2001), s. 2054-2056 ISSN 0892-6638 R&D Projects: GA ČR GA204/00/1253 Institutional research plan: CEZ:AV0Z5020903 Keywords : prokaryotic development Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 8.817, year: 2001

[A study of recombinant human sestrin 1 and sestrin 2 proteins produced in a prokaryotic system].

Science.gov (United States)

Rai, N; Kumar, R; Haque, Md A; Hassan, Md I; Dey, S

2017-01-01

Sestrins are highly conserved stress-inducible proteins capable of suppressing the production of ROS and signalling through mTORC1. Here we report a study of human sestrin1 (sesn1) and sestrin2 (sesn2) proteins produced in a pET28^(+) vector based prokaryotic system. Mass spectrometry analysis, western blot and surface plasmon resonance (SPR) of affinity purified sesn1 and sesn2 proteins confirmed their identity; biophysical characteristics were observed using circular dichroism (CD) showing that sesn1 and sesn2 have a predominant α-helical structure. Here we describe a simple, one step purification process to purify a large amount of sestrin proteins with significant yield. Further study of recombinant human sestrins may further facilitate the understanding of their roles in eukaryotic cells.

Alphavirus Replicon DNA Vectors Expressing Ebola GP and VP40 Antigens Induce Humoral and Cellular Immune Responses in Mice

Directory of Open Access Journals (Sweden)

Shoufeng Ren

2018-01-01

Full Text Available Ebola virus (EBOV causes severe hemorrhagic fevers in humans, and no approved therapeutics or vaccine is currently available. Glycoprotein (GP is the major protective antigen of EBOV, and can generate virus-like particles (VLPs by co-expression with matrix protein (VP40. In this study, we constructed a recombinant Alphavirus Semliki Forest virus (SFV replicon vector DREP to express EBOV GP and matrix viral protein (VP40. EBOV VLPs were successfully generated and achieved budding from 293 cells after co-transfection with DREP-based GP and VP40 vectors (DREP-GP+DREP-VP40. Vaccination of BALB/c mice with DREP-GP, DREP-VP40, or DREP-GP+DREP-VP40 vectors, followed by immediate electroporation resulted in a mixed IgG subclass production, which recognized EBOV GP and/or VP40 proteins. This vaccination regimen also led to the generation of both Th1 and Th2 cellular immune responses in mice. Notably, vaccination with DREP-GP and DREP-VP40, which produces both GP and VP40 antigens, induced a significantly higher level of anti-GP IgG2a antibody and increased IFN-γ secreting CD8+ T-cell responses relative to vaccination with DREP-GP or DREP-VP40 vector alone. Our study indicates that co-expression of GP and VP40 antigens based on the SFV replicon vector generates EBOV VLPs in vitro, and vaccination with recombinant DREP vectors containing GP and VP40 antigens induces Ebola antigen-specific humoral and cellular immune responses in mice. This novel approach provides a simple and efficient vaccine platform for Ebola disease prevention.

Recombinant vaccines against T. gondii: comparison between homologous and heterologous vaccination protocols using two viral vectors expressing SAG1.

Science.gov (United States)

Mendes, Érica Araújo; Fonseca, Flavio G; Casério, Bárbara M; Colina, Janaína P; Gazzinelli, Ricardo Tostes; Caetano, Braulia C

2013-01-01

The use of recombinant viral vectors expressing T. gondii antigens is a safe and efficient approach to induce immune response against the parasite and a valuable tool for vaccine development. We have previously protected mice from toxoplasmosis by immunizing the animals with an adenovirus expressing the protein SAG1 (AdSAG1) of T. gondii. We are now looking for ways to improve the vaccination strategy and enhance protection. One limitation of homologous vaccinations (sequential doses of the same vector) is induction of anti-vector immune response that blocks cell transduction, restricts transgene expression and, consequently, compromises the overall outcome of vaccination. One way to avert the effects of anti-vector response is to use different viruses in prime and boost (heterologous vaccination). Bearing this in mind, we generated a modified Vaccinia Virus Ankara encoding SAG1 (MVASAG1), to be tested as boost agent after prime with AdSAG1. Although minor differences were observed in the magnitude of the anti-SAG1 immune response induced by each vaccination protocol, the heterologous immunization with AdSAG1 followed by MVASAG1 resulted in improved capacity to control brain cyst formation in a model of chronic toxoplasmosis in C57BL/6 mice.

Recombinant vaccines against T. gondii: comparison between homologous and heterologous vaccination protocols using two viral vectors expressing SAG1.

Directory of Open Access Journals (Sweden)

Érica Araújo Mendes

Full Text Available The use of recombinant viral vectors expressing T. gondii antigens is a safe and efficient approach to induce immune response against the parasite and a valuable tool for vaccine development. We have previously protected mice from toxoplasmosis by immunizing the animals with an adenovirus expressing the protein SAG1 (AdSAG1 of T. gondii. We are now looking for ways to improve the vaccination strategy and enhance protection. One limitation of homologous vaccinations (sequential doses of the same vector is induction of anti-vector immune response that blocks cell transduction, restricts transgene expression and, consequently, compromises the overall outcome of vaccination. One way to avert the effects of anti-vector response is to use different viruses in prime and boost (heterologous vaccination. Bearing this in mind, we generated a modified Vaccinia Virus Ankara encoding SAG1 (MVASAG1, to be tested as boost agent after prime with AdSAG1. Although minor differences were observed in the magnitude of the anti-SAG1 immune response induced by each vaccination protocol, the heterologous immunization with AdSAG1 followed by MVASAG1 resulted in improved capacity to control brain cyst formation in a model of chronic toxoplasmosis in C57BL/6 mice.

Evolution and Diversity of the Ras Superfamily of Small GTPases in Prokaryotes

Science.gov (United States)

Wuichet, Kristin; Søgaard-Andersen, Lotte

2015-01-01

The Ras superfamily of small GTPases are single domain nucleotide-dependent molecular switches that act as highly tuned regulators of complex signal transduction pathways. Originally identified in eukaryotes for their roles in fundamental cellular processes including proliferation, motility, polarity, nuclear transport, and vesicle transport, recent studies have revealed that single domain GTPases also control complex functions such as cell polarity, motility, predation, development and antibiotic resistance in bacteria. Here, we used a computational genomics approach to understand the abundance, diversity, and evolution of small GTPases in prokaryotes. We collected 520 small GTPase sequences present in 17% of 1,611 prokaryotic genomes analyzed that cover diverse lineages. We identified two discrete families of small GTPases in prokaryotes that show evidence of three distinct catalytic mechanisms. The MglA family includes MglA homologs, which are typically associated with the MglB GTPase activating protein, whereas members of the Rup (Ras superfamily GTPase of unknown function in prokaryotes) family are not predicted to interact with MglB homologs. System classification and genome context analyses support the involvement of small GTPases in diverse prokaryotic signal transduction pathways including two component systems, laying the foundation for future experimental characterization of these proteins. Phylogenetic analysis of prokaryotic and eukaryotic GTPases supports that the last universal common ancestor contained ancestral MglA and Rup family members. We propose that the MglA family was lost from the ancestral eukaryote and that the Ras superfamily members in extant eukaryotes are the result of vertical and horizontal gene transfer events of ancestral Rup GTPases. PMID:25480683

How crowded is the prokaryotic cytoplasm?

NARCIS (Netherlands)

Spitzer, Jan; Poolman, Bert; Ferguson, Stuart

2013-01-01

We consider biomacromolecular crowding within the cytoplasm of prokaryotic cells as a two-phase system of 'supercrowded' cytogel and 'dilute' cytosol; we simplify and quantify this model for a coccoid cell over a wide range of biomacromolecular crowding. The key result shows that the supercrowded

Getting out : protein traffic in prokaryotes

NARCIS (Netherlands)

Pugsley, A.P; Francetic, O; Driessen, A.J.M.; de Lorenzo, V.

Protein secretion systems in prokaryotes are increasingly shifting from being considered as experimental models for 'more complex' processes (i.e. eukaryotes) to being a major source of key biological questions in their own right. The pathways by which proteins move between compartments or insert

Enhancing poxvirus vectors vaccine immunogenicity.

Science.gov (United States)

García-Arriaza, Juan; Esteban, Mariano

2014-01-01

Attenuated recombinant poxvirus vectors expressing heterologous antigens from pathogens are currently at various stages in clinical trials with the aim to establish their efficacy. This is because these vectors have shown excellent safety profiles, significant immunogenicity against foreign expressed antigens and are able to induce protective immune responses. In view of the limited efficacy triggered by some poxvirus strains used in clinical trials (i.e, ALVAC in the RV144 phase III clinical trial for HIV), and of the restrictive replication capacity of the highly attenuated vectors like MVA and NYVAC, there is a consensus that further improvements of these vectors should be pursuit. In this review we considered several strategies that are currently being implemented, as well as new approaches, to improve the immunogenicity of the poxvirus vectors. This includes heterologous prime/boost protocols, use of co-stimulatory molecules, deletion of viral immunomodulatory genes still present in the poxvirus genome, enhancing virus promoter strength, enhancing vector replication capacity, optimizing expression of foreign heterologous sequences, and the combined use of adjuvants. An optimized poxvirus vector triggering long-lasting immunity with a high protective efficacy against a selective disease should be sought.

Geochemical Interactions and Viral-Prokaryote Relationships in Freshwater Environments

Science.gov (United States)

Kyle, J. E.; Ferris, G.

2009-05-01

Viral and prokaryotic abundances were surveyed throughout southern Ontario aquatic habitats to determine relationships with geochemical parameters in the natural environment. Surface water samples were collected from acid mine drainage in summer of 2007 and 2008 and from circum-neutral pH environments in October to November 2008. Site determination was based on collecting samples from various aquatic habitats (acid mine drainage, lakes, rivers, tributaries, wetlands) with differing bedrock geology (limestone and shale dominated vs granitic Canadian Shield) to obtain a range of geochemical conditions. At each site, measurements of temperature, pH, and Eh were conducted. Samples collected for microbial counts and electron imaging were preserved to a final concentration of 2.5 % (v/v) glutaraldehyde. Additional sample were filtered into 60 mL nalgene bottles and amber EPA certified 40 mL glass vials to determine chemical constituents and dissolved organic carbon (DOC), respectively. Water was also collected to determine additional physiochemical parameters (dissolved total iron, ferric iron, nitrate, sulfate, phosphate, alkalinity, and turbidity). All samples were stored at 4 °C until analysis. Viral and prokaryotic abundance was determined by staining samples with SYBR Green I and examining with a epifluorescence microscope under blue excitation. Multiple regression analysis using stepwise backwards regression and general linear models revealed that viral abundance was the most influential predictor of prokaryotic abundance. Additional predictors include pH, sulfate, phosphate, and magnesium. The strength of the model was very strong with 90 % of the variability explained (R2 = 0.90, p < 0.007). This is the first report, to our knowledge, of viruses exhibiting such strong controls over prokaryotic abundance in the natural environment. All relationships are positively correlated with the exception of Mg, which is negatively correlated. Iron was also noted as a

A common multiple cloning site in a set of vectors for expression of eukaryotic genes in mammalian, insect and bacterial cells

DEFF Research Database (Denmark)

Pallisgaard, N; Pedersen, FS; Birkelund, Svend

1994-01-01

a start Met codon was included in the same reading frame as in lambda gt11Sfi-Not to support expression of partial cDNA clones. Thus a cDNA insert of lambda gt11Sfi-Not could be shuttled among the new vectors for expression. The other set of vectors without a start codon were suitable for expression of c......DNA carrying their own start Met codon. By Western blot analysis and by transactivation of a reporter plasmid in co-transfections we show that cDNA is very efficiently expressed in NIH 3T3 cells under control of the elongation factor 1 alpha promoter....

A human parvovirus, adeno-associated virus, as a eucaryotic vector: Transient expression and encapsidation of the procaryotic gene for chloramphenicol acetyltransferase

Energy Technology Data Exchange (ETDEWEB)

Tratschin, J.D.; West, M.H.P.; Sandbank, T.; Carter, B.J.

1984-10-01

The authors have used the defective human parvovirus adeno-associated virus (AAV) as a novel eurocaryotic vector (parvector) for the expression of a foreign gene in human cells. The recombinant, pAV2, contains the AAV genome in a pBR322-derived bacterial plasmid. When pAV2 is transfected into human cells together with helper adenovirus particles, the AAV genome is rescued from the recombinant plasmid and replicated to produce infectious AAV particles at high efficiency. To create a vector, we inserted a procaryotic sequence coding for chloramphenicol acetyltransferase (CAT) into derivatives of pAV2 following either of the AAV promoters p/sub 40/ (pAVHiCAT) and p/sub 19/ (pAVBcCAT). When transfected into human 293 cells or HeLa cells, pAVHiCAT expressed CAT activity in the absence of adenovirus. In the presence of adenovirus, this vector produced increased amounts of CAT activity and the recombinant AAV-CAT genome was replicated. In 293 cells, pAVBcCAT expressed a similar amount of CAT activity in the absence or presence of adenovirus and the recombinant AAV-CAT genome was not replicated. In HeLa cells, pAVBcCAT expressed low levels of CAT activity, but this level was elevated by coinfection with adenovirus particles or by cotransfection with a plasmid which expressed the adenovirus early region 1A (E1A) product. The E1A product is a transcriptional activator and is expressed in 293 cells. Thus, expression from two AAV promoters is differentially regulated: expression from p/sub 19/ is increased by E1A, whereas p/sub 40/ yields high levels of constitutive expression in the absence of E1A. Both AAV vectors were packaged into AAV particles by complementation with wild-type AAV and yielded CAT activity when subsequently infected into cells in the presence of adenovirus.

Global identification of prokaryotic glycoproteins based on an Escherichia coli proteome microarray.

Directory of Open Access Journals (Sweden)

Zong-Xiu Wang

Full Text Available Glycosylation is one of the most abundant protein posttranslational modifications. Protein glycosylation plays important roles not only in eukaryotes but also in prokaryotes. To further understand the roles of protein glycosylation in prokaryotes, we developed a lectin binding assay to screen glycoproteins on an Escherichia coli proteome microarray containing 4,256 affinity-purified E.coli proteins. Twenty-three E.coli proteins that bound Wheat-Germ Agglutinin (WGA were identified. PANTHER protein classification analysis showed that these glycoprotein candidates were highly enriched in metabolic process and catalytic activity classes. One sub-network centered on deoxyribonuclease I (sbcB was identified. Bioinformatics analysis suggests that prokaryotic protein glycosylation may play roles in nucleotide and nucleic acid metabolism. Fifteen of the 23 glycoprotein candidates were validated by lectin (WGA staining, thereby increasing the number of validated E. coli glycoproteins from 3 to 18. By cataloguing glycoproteins in E.coli, our study greatly extends our understanding of protein glycosylation in prokaryotes.

PSSRdb: a relational database of polymorphic simple sequence repeats extracted from prokaryotic genomes.

Science.gov (United States)

Kumar, Pankaj; Chaitanya, Pasumarthy S; Nagarajaram, Hampapathalu A

2011-01-01

PSSRdb (Polymorphic Simple Sequence Repeats database) (http://www.cdfd.org.in/PSSRdb/) is a relational database of polymorphic simple sequence repeats (PSSRs) extracted from 85 different species of prokaryotes. Simple sequence repeats (SSRs) are the tandem repeats of nucleotide motifs of the sizes 1-6 bp and are highly polymorphic. SSR mutations in and around coding regions affect transcription and translation of genes. Such changes underpin phase variations and antigenic variations seen in some bacteria. Although SSR-mediated phase variation and antigenic variations have been well-studied in some bacteria there seems a lot of other species of prokaryotes yet to be investigated for SSR mediated adaptive and other evolutionary advantages. As a part of our on-going studies on SSR polymorphism in prokaryotes we compared the genome sequences of various strains and isolates available for 85 different species of prokaryotes and extracted a number of SSRs showing length variations and created a relational database called PSSRdb. This database gives useful information such as location of PSSRs in genomes, length variation across genomes, the regions harboring PSSRs, etc. The information provided in this database is very useful for further research and analysis of SSRs in prokaryotes.

Two Tales of Prokaryotic Genomic Diversity: Escherichia coli and Halophiles

Directory of Open Access Journals (Sweden)

Lejla Pašić

2014-01-01

Full Text Available Prokaryotes are generally characterized by vast genomic diversity that has been shaped by mutations, horizontal gene transfer, bacteriocins and phage predation. Enormous genetic diversity has developed as a result of stresses imposed in harsh environments and the ability of microorganisms to adapt. Two examples of prokaryotic diversity are presented: on intraspecies level, exemplified by Escherichia coli, and the diversity of the hypersaline environment, with the discussion of food-related health issues and biotechnological potential.

Prokaryotic cells: structural organisation of the cytoskeleton and organelles

Directory of Open Access Journals (Sweden)

Wanderley de Souza

2012-05-01

Full Text Available For many years, prokaryotic cells were distinguished from eukaryotic cells based on the simplicity of their cytoplasm, in which the presence of organelles and cytoskeletal structures had not been discovered. Based on current knowledge, this review describes the complex components of the prokaryotic cell cytoskeleton, including (i tubulin homologues composed of FtsZ, BtuA, BtuB and several associated proteins, which play a fundamental role in cell division, (ii actin-like homologues, such as MreB and Mb1, which are involved in controlling cell width and cell length, and (iii intermediate filament homologues, including crescentin and CfpA, which localise on the concave side of a bacterium and along its inner curvature and associate with its membrane. Some prokaryotes exhibit specialised membrane-bound organelles in the cytoplasm, such as magnetosomes and acidocalcisomes, as well as protein complexes, such as carboxysomes. This review also examines recent data on the presence of nanotubes, which are structures that are well characterised in mammalian cells that allow direct contact and communication between cells.

Gene duplications in prokaryotes can be associated with environmental adaptation.

Science.gov (United States)

Bratlie, Marit S; Johansen, Jostein; Sherman, Brad T; Huang, Da Wei; Lempicki, Richard A; Drabløs, Finn

2010-10-20

Gene duplication is a normal evolutionary process. If there is no selective advantage in keeping the duplicated gene, it is usually reduced to a pseudogene and disappears from the genome. However, some paralogs are retained. These gene products are likely to be beneficial to the organism, e.g. in adaptation to new environmental conditions. The aim of our analysis is to investigate the properties of paralog-forming genes in prokaryotes, and to analyse the role of these retained paralogs by relating gene properties to life style of the corresponding prokaryotes. Paralogs were identified in a number of prokaryotes, and these paralogs were compared to singletons of persistent orthologs based on functional classification. This showed that the paralogs were associated with for example energy production, cell motility, ion transport, and defence mechanisms. A statistical overrepresentation analysis of gene and protein annotations was based on paralogs of the 200 prokaryotes with the highest fraction of paralog-forming genes. Biclustering of overrepresented gene ontology terms versus species was used to identify clusters of properties associated with clusters of species. The clusters were classified using similarity scores on properties and species to identify interesting clusters, and a subset of clusters were analysed by comparison to literature data. This analysis showed that paralogs often are associated with properties that are important for survival and proliferation of the specific organisms. This includes processes like ion transport, locomotion, chemotaxis and photosynthesis. However, the analysis also showed that the gene ontology terms sometimes were too general, imprecise or even misleading for automatic analysis. Properties described by gene ontology terms identified in the overrepresentation analysis are often consistent with individual prokaryote lifestyles and are likely to give a competitive advantage to the organism. Paralogs and singletons dominate

Metabolic profiles of prokaryotic and eukaryotic communities in deep-sea sponge Neamphius huxleyi indicated by metagenomics

Science.gov (United States)

Li, Zhi-Yong; Wang, Yue-Zhu; He, Li-Ming; Zheng, Hua-Jun

2014-01-01

The whole metabolism of a sponge holobiont and the respective contributions of prokaryotic and eukaryotic symbionts and their associations with the sponge host remain largely unclear. Meanwhile, compared with shallow water sponges, deep-sea sponges are rarely understood. Here we report the metagenomic exploration of deep-sea sponge Neamphius huxleyi at the whole community level. Metagenomic data showed phylogenetically diverse prokaryotes and eukaryotes in Neamphius huxleyi. MEGAN and gene enrichment analyses indicated different metabolic potentials of prokaryotic symbionts from eukaryotic symbionts, especially in nitrogen and carbon metabolisms, and their molecular interactions with the sponge host. These results supported the hypothesis that prokaryotic and eukaryotic symbionts have different ecological roles and relationships with sponge host. Moreover, vigorous denitrification, and CO2 fixation by chemoautotrophic prokaryotes were suggested for this deep-sea sponge. The study provided novel insights into the respective potentials of prokaryotic and eukaryotic symbionts and their associations with deep-sea sponge Neamphius huxleyi. PMID:24463735

Metabolic profiles of prokaryotic and eukaryotic communities in deep-sea sponge Lamellomorpha sp. indicated by metagenomics

Science.gov (United States)

Li, Zhi-Yong; Wang, Yue-Zhu; He, Li-Ming; Zheng, Hua-Jun

2014-01-01

The whole metabolism of a sponge holobiont and the respective contributions of prokaryotic and eukaryotic symbionts and their associations with the sponge host remain largely unclear. Meanwhile, compared with shallow water sponges, deep-sea sponges are rarely understood. Here we report the metagenomic exploration of deep-sea sponge Lamellomorpha sp. at the whole community level. Metagenomic data showed phylogenetically diverse prokaryotes and eukaryotes in Lamellomorpha sp.. MEGAN and gene enrichment analyses indicated different metabolic potentials of prokaryotic symbionts from eukaryotic symbionts, especially in nitrogen and carbon metabolisms, and their molecular interactions with the sponge host. These results supported the hypothesis that prokaryotic and eukaryotic symbionts have different ecological roles and relationships with sponge host. Moreover, vigorous denitrification, and CO2 fixation by chemoautotrophic prokaryotes were suggested for this deep-sea sponge. The study provided novel insights into the respective potentials of prokaryotic and eukaryotic symbionts and their associations with deep-sea sponge Lamellomorpha sp..

Metabolic Compensation and Circadian Resilience in Prokaryotic Cyanobacteria

Science.gov (United States)

Johnson, Carl Hirschie; Egli, Martin

2014-01-01

For a biological oscillator to function as a circadian pacemaker that confers a fitness advantage, its timing functions must be stable in response to environmental and metabolic fluctuations. One such stability enhancer, temperature compensation, has long been a defining characteristic of these timekeepers. However, an accurate biological timekeeper must also resist changes in metabolism, and this review suggests that temperature compensation is actually a subset of a larger phenomenon, namely metabolic compensation, which maintains the frequency of circadian oscillators in response to a host of factors that impinge on metabolism and would otherwise destabilize these clocks. The circadian system of prokaryotic cyanobacteria is an illustrative model because it is composed of transcriptional and nontranscriptional oscillators that are coupled to promote resilience. Moreover, the cyanobacterial circadian program regulates gene activity and metabolic pathways, and it can be manipulated to improve the expression of bioproducts that have practical value. PMID:24905782

Keeping the wolves at bay: antitoxins of prokaryotic type II toxin-antitoxin systems

Directory of Open Access Journals (Sweden)

Wai Ting eChan

2016-03-01

Full Text Available In their initial stages of discovery, prokaryotic toxin-antitoxin (TA systems were confined to bacterial plasmids where they function to mediate the maintenance and stability of usually low- to medium-copy number plasmids through the post-segregational killing of any plasmid-free daughter cells that developed. Their eventual discovery as nearly ubiquitous and repetitive elements in bacterial chromosomes led to a wealth of knowledge and scientific debate as to their diversity and functionality in the prokaryotic lifestyle. Currently categorized into six different types designated types I – VI, type II TA systems are the best characterized. These generally comprised of two genes encoding a proteic toxin and its corresponding proteic antitoxin, respectively. Under normal growth conditions, the stable toxin is prevented from exerting its lethal effect through tight binding with the less stable antitoxin partner, forming a non-lethal TA protein complex. Besides binding with its cognate toxin, the antitoxin also plays a role in regulating the expression of the type II TA operon by binding to the operator site, thereby repressing transcription from the TA promoter. In most cases, full repression is observed in the presence of the TA complex as binding of the toxin enhances the DNA binding capability of the antitoxin. TA systems have been implicated in a gamut of prokaryotic cellular functions such as being mediators of programmed cell death as well as persistence or dormancy, biofilm formation, as defensive weapons against bacteriophage infections and as virulence factors in pathogenic bacteria. It is thus apparent that these antitoxins, as DNA-binding proteins, play an essential role in modulating the prokaryotic lifestyle whilst at the same time preventing the lethal action of the toxins under normal growth conditions, i.e., keeping the proverbial wolves at bay. In this review, we will cover the diversity and characteristics of various type II TA

Diversity and activity in marine prokaryotes

NARCIS (Netherlands)

Arrieta López de Uralde, Jesús Maria

2005-01-01

Life on Earth epends on the endless recycling of elements as matter and energy are required to sustain life. The prokaryotes (Bacteria and Archaea) are the masters of the trade of life. After all, they were already responsible for the major biogeochemical cycles 3.000 million years ago, long before

Microscopic Identification of Prokaryotes in Modern and Ancient Halite, Saline Valley and Death Valley, California

Science.gov (United States)

Schubert, Brian A.; Lowenstein, Tim K.; Timofeeff, Michael N.

2009-06-01

Primary fluid inclusions in halite crystallized in Saline Valley, California, in 1980, 2004-2005, and 2007, contain rod- and coccoid-shaped microparticles the same size and morphology as archaea and bacteria living in modern brines. Primary fluid inclusions from a well-dated (0-100,000 years), 90 m long salt core from Badwater Basin, Death Valley, California, also contain microparticles, here interpreted as halophilic and halotolerant prokaryotes. Prokaryotes are distinguished from crystals on the basis of morphology, optical properties (birefringence), and uniformity of size. Electron micrographs of microparticles from filtered modern brine (Saline Valley), dissolved modern halite crystals (Saline Valley), and dissolved ancient halite crystals (Death Valley) support in situ microscopic observations that prokaryotes are present in fluid inclusions in ancient halite. In the Death Valley salt core, prokaryotes in fluid inclusions occur almost exclusively in halite precipitated in perennial saline lakes 10,000 to 35,000 years ago. This suggests that trapping and preservation of prokaryotes in fluid inclusions is influenced by the surface environment in which the halite originally precipitated. In all cases, prokaryotes in fluid inclusions in halite from the Death Valley salt core are miniaturized (<1 μm diameter cocci, <2.5 μm long, very rare rod shapes), which supports interpretations that the prokaryotes are indigenous to the halite and starvation survival may be the normal response of some prokaryotes to entrapment in fluid inclusions for millennia. These results reinforce the view that fluid inclusions in halite and possibly other evaporites are important repositories of microbial life and should be carefully examined in the search for ancient microorganisms on Earth, Mars, and elsewhere in the Solar System.

Modeling the winter-to-summer transition of prokaryotic and viral abundance in the Arctic Ocean.

Science.gov (United States)

Winter, Christian; Payet, Jérôme P; Suttle, Curtis A

2012-01-01

One of the challenges in oceanography is to understand the influence of environmental factors on the abundances of prokaryotes and viruses. Generally, conventional statistical methods resolve trends well, but more complex relationships are difficult to explore. In such cases, Artificial Neural Networks (ANNs) offer an alternative way for data analysis. Here, we developed ANN-based models of prokaryotic and viral abundances in the Arctic Ocean. The models were used to identify the best predictors for prokaryotic and viral abundances including cytometrically-distinguishable populations of prokaryotes (high and low nucleic acid cells) and viruses (high- and low-fluorescent viruses) among salinity, temperature, depth, day length, and the concentration of Chlorophyll-a. The best performing ANNs to model the abundances of high and low nucleic acid cells used temperature and Chl-a as input parameters, while the abundances of high- and low-fluorescent viruses used depth, Chl-a, and day length as input parameters. Decreasing viral abundance with increasing depth and decreasing system productivity was captured well by the ANNs. Despite identifying the same predictors for the two populations of prokaryotes and viruses, respectively, the structure of the best performing ANNs differed between high and low nucleic acid cells and between high- and low-fluorescent viruses. Also, the two prokaryotic and viral groups responded differently to changes in the predictor parameters; hence, the cytometric distinction between these populations is ecologically relevant. The models imply that temperature is the main factor explaining most of the variation in the abundances of high nucleic acid cells and total prokaryotes and that the mechanisms governing the reaction to changes in the environment are distinctly different among the prokaryotic and viral populations.

Modeling the Winter–to–Summer Transition of Prokaryotic and Viral Abundance in the Arctic Ocean

Science.gov (United States)

Winter, Christian; Payet, Jérôme P.; Suttle, Curtis A.

2012-01-01

One of the challenges in oceanography is to understand the influence of environmental factors on the abundances of prokaryotes and viruses. Generally, conventional statistical methods resolve trends well, but more complex relationships are difficult to explore. In such cases, Artificial Neural Networks (ANNs) offer an alternative way for data analysis. Here, we developed ANN-based models of prokaryotic and viral abundances in the Arctic Ocean. The models were used to identify the best predictors for prokaryotic and viral abundances including cytometrically-distinguishable populations of prokaryotes (high and low nucleic acid cells) and viruses (high- and low-fluorescent viruses) among salinity, temperature, depth, day length, and the concentration of Chlorophyll-a. The best performing ANNs to model the abundances of high and low nucleic acid cells used temperature and Chl-a as input parameters, while the abundances of high- and low-fluorescent viruses used depth, Chl-a, and day length as input parameters. Decreasing viral abundance with increasing depth and decreasing system productivity was captured well by the ANNs. Despite identifying the same predictors for the two populations of prokaryotes and viruses, respectively, the structure of the best performing ANNs differed between high and low nucleic acid cells and between high- and low-fluorescent viruses. Also, the two prokaryotic and viral groups responded differently to changes in the predictor parameters; hence, the cytometric distinction between these populations is ecologically relevant. The models imply that temperature is the main factor explaining most of the variation in the abundances of high nucleic acid cells and total prokaryotes and that the mechanisms governing the reaction to changes in the environment are distinctly different among the prokaryotic and viral populations. PMID:23285186

Efficient delivery and stable gene expression in a hematopoietic cell line using a chimeric serotype 35 fiber pseudotyped helper-dependent adenoviral vector

International Nuclear Information System (INIS)

Balamotis, Michael Andrew; Huang, Katie; Mitani, Kohnosuke

2004-01-01

Certain human cell populations have remained difficult to infect with human adenovirus (Ad) serotype 5 because of their lack of coxsackievirus B-adenovirus receptor (CAR). Native adenovirus fiber compositions, although diverse, cannot infect all tissue types. Recently, a chimeric Ad5/35 fiber was created, which displays an altered tropism from Ad5. We incorporated this chimeric fiber into a helper-dependent (HD) adenovirus vector system and compared HD to E1-deleted (E1Δ) vectors by transgene expression, cell transduction efficiency, and cytotoxicity. K562 cells were infected ∼50 times more efficiently with the chimeric Ad5/35 fiber compared with the Ad5 fiber. Short-term transgene expression was sustained longer from HD Ad5/35 than E1Δ Ad5/35 vector after in vitro infection of actively dividing K562 cells. Rapid loss of transgene expression from E1Δ Ad5/35 infection was not due to the loss of vector genomes, as determined by quantitative real-time PCR (QRT-PCR), or cytotoxicity, but rather through a putative silencing mechanism

Construction and expression of secreting type human TRAIL gene vector mediated by hypoxia/radiation double sensitive promoter

International Nuclear Information System (INIS)

Yang Yanming; Jia Xiaojing; Qu Yaqin; Li Yanbo

2009-01-01

Objective: To construct secreting type human TRAIL (shTRAIL) gene vector pcDNA3.1-HRE/Egr1-shTRAIL mediated by hypoxia/radiation double sensitive promoter, and observe the effect of hypoxia and radiation on shTRAIL. Methods: HRE upper and lower strands were gotten by chemical synthesis, double strands HRE was gotten by PCR; pMD19T-Egr1 was digested by Sac I and Hind III, then Egr1 was obtained, pshuttle-shTRAIL was digested by Kpn I and BamH I, then shTRAIL was obtained; HRE/Egr1 double sensitive promoter mediated shTRAIL expression vector pcDNA3.1-HRE/Egr1-shTRAIL was constructed by gene recombination technique, it was identified correctly by enzyme digestion, PCR and sequencing. A549 cells were divided into normal, hypoxia (0.1%), irradiation (6 Gy) and hypoxia + irradiation groups. Results: After enzyme digestion by BamH I and Sma I, the fragments which lengths were 1284 bp and 4 998 bp, 2 292 bp and 3 990 bp were obtained; the vector was amplified by PCR with Egr1 and shTRAIL primer, the products which lengthens were 469 bp and 820 bp were obtained; pcDNA3.1-HRE/Egr1-shTRAIL was sequenced, the result was same to designed, this demonstrated that the construction was right. The vectors were transfected into A549 cells of adenocarcinoma of lung, the expression levels of shTRAIL mRNA and protein were increased after treated with hypoxia and radiation, it had statistically significant differences compared with normal group (P<0.05), and when they were combinated, the effect was more obvious. Conclusion: Secreting type human TRAIL gene vector pcDNA3.1-HRE/Egr1-shTRAIL mediated by hypoxia/radiation double sensitive promoter is constructed successfully, and hypoxia and radiation could increase the expression of TRAIL, and they have synergetic effect. (authors)

A plasmid toolkit for cloning chimeric cDNAs encoding customized fusion proteins into any Gateway destination expression vector

Science.gov (United States)

2013-01-01

Background Valuable clone collections encoding the complete ORFeomes for some model organisms have been constructed following the completion of their genome sequencing projects. These libraries are based on Gateway cloning technology, which facilitates the study of protein function by simplifying the subcloning of open reading frames (ORF) into any suitable destination vector. The expression of proteins of interest as fusions with functional modules is a frequent approach in their initial functional characterization. A limited number of Gateway destination expression vectors allow the construction of fusion proteins from ORFeome-derived sequences, but they are restricted to the possibilities offered by their inbuilt functional modules and their pre-defined model organism-specificity. Thus, the availability of cloning systems that overcome these limitations would be highly advantageous. Results We present a versatile cloning toolkit for constructing fully-customizable three-part fusion proteins based on the MultiSite Gateway cloning system. The fusion protein components are encoded in the three plasmids integral to the kit. These can recombine with any purposely-engineered destination vector that uses a heterologous promoter external to the Gateway cassette, leading to the in-frame cloning of an ORF of interest flanked by two functional modules. In contrast to previous systems, a third part becomes available for peptide-encoding as it no longer needs to contain a promoter, resulting in an increased number of possible fusion combinations. We have constructed the kit’s component plasmids and demonstrate its functionality by providing proof-of-principle data on the expression of prototype fluorescent fusions in transiently-transfected cells. Conclusions We have developed a toolkit for creating fusion proteins with customized N- and C-term modules from Gateway entry clones encoding ORFs of interest. Importantly, our method allows entry clones obtained from ORFeome

Gauge anomaly with vector and axial-vector fields in 6D curved space

Science.gov (United States)

Yajima, Satoshi; Eguchi, Kohei; Fukuda, Makoto; Oka, Tomonori

2018-03-01

Imposing the conservation equation of the vector current for a fermion of spin 1/2 at the quantum level, a gauge anomaly for the fermion coupling with non-Abelian vector and axial-vector fields in 6D curved space is expressed in tensorial form. The anomaly consists of terms that resemble the chiral U(1) anomaly and the commutator terms that disappear if the axial-vector field is Abelian.

Heterogeneous distribution of prokaryotes and viruses at the microscale in a tidal sediment

DEFF Research Database (Denmark)

Carreira, Cátia; Larsen, Morten; Glud, Ronnie

2013-01-01

In this study we show for the first time the microscale (mm) 2- and 3-dimensional spatial distribution and abundance of prokaryotes, viruses, and oxygen in a tidal sediment. Prokaryotes and viruses were highly heterogeneously distributed with patches of elevated abundances surrounded by areas of ...

Vector regression introduced

Directory of Open Access Journals (Sweden)

Mok Tik

2014-06-01

Full Text Available This study formulates regression of vector data that will enable statistical analysis of various geodetic phenomena such as, polar motion, ocean currents, typhoon/hurricane tracking, crustal deformations, and precursory earthquake signals. The observed vector variable of an event (dependent vector variable is expressed as a function of a number of hypothesized phenomena realized also as vector variables (independent vector variables and/or scalar variables that are likely to impact the dependent vector variable. The proposed representation has the unique property of solving the coefficients of independent vector variables (explanatory variables also as vectors, hence it supersedes multivariate multiple regression models, in which the unknown coefficients are scalar quantities. For the solution, complex numbers are used to rep- resent vector information, and the method of least squares is deployed to estimate the vector model parameters after transforming the complex vector regression model into a real vector regression model through isomorphism. Various operational statistics for testing the predictive significance of the estimated vector parameter coefficients are also derived. A simple numerical example demonstrates the use of the proposed vector regression analysis in modeling typhoon paths.

Immunogenicity of ORFV-based vectors expressing the rabies virus glycoprotein in livestock species.

Science.gov (United States)

Martins, Mathias; Joshi, Lok R; Rodrigues, Fernando S; Anziliero, Deniz; Frandoloso, Rafael; Kutish, Gerald F; Rock, Daniel L; Weiblen, Rudi; Flores, Eduardo F; Diel, Diego G

2017-11-01

The parapoxvirus Orf virus (ORFV) encodes several immunomodulatory proteins (IMPs) that modulate host-innate and pro-inflammatory responses and has been proposed as a vaccine delivery vector for use in animal species. Here we describe the construction and characterization of two recombinant ORFV vectors expressing the rabies virus (RABV) glycoprotein (G). The RABV-G gene was inserted in the ORFV024 or ORFV121 gene loci, which encode for IMPs that are unique to parapoxviruses and inhibit activation of the NF-κB signaling pathway. The immunogenicity of the resultant recombinant viruses (ORFV ∆024 RABV-G or ORFV ∆121 RABV-G, respectively) was evaluated in pigs and cattle. Immunization of the target species with ORFV ∆024 RABV-G and ORFV ∆121 RABV-G elicited robust neutralizing antibody responses against RABV. Notably, neutralizing antibody titers induced in ORFV ∆121 RABV-G-immunized pigs and cattle were significantly higher than those detected in ORFV ∆024 RABV-G-immunized animals, indicating a higher immunogenicity of ORFV Δ121 -based vectors in these animal species. Copyright © 2017 Elsevier Inc. All rights reserved.

Neonatal Gene Therapy for Hemophilia B by a Novel Adenovirus Vector Showing Reduced Leaky Expression of Viral Genes.

Science.gov (United States)

Iizuka, Shunsuke; Sakurai, Fuminori; Tachibana, Masashi; Ohashi, Kazuo; Mizuguchi, Hiroyuki

2017-09-15

Gene therapy during neonatal and infant stages is a promising approach for hemophilia B, a congenital disorder caused by deficiency of blood coagulation factor IX (FIX). An adenovirus (Ad) vector has high potential for use in neonatal or infant gene therapy for hemophilia B due to its superior transduction properties; however, leaky expression of Ad genes often reduces the transduction efficiencies by Ad protein-mediated tissue damage. Here, we used a novel Ad vector, Ad-E4-122aT, which exhibits a reduction in the leaky expression of Ad genes in liver, in gene therapy studies for neonatal hemophilia B mice. Ad-E4-122aT exhibited significantly higher transduction efficiencies than a conventional Ad vector in neonatal mice. In neonatal hemophilia B mice, a single neonatal injection of Ad-E4-122aT expressing human FIX (hFIX) (Ad-E4-122aT-AHAFIX) maintained more than 6% of the normal plasma hFIX activity levels for approximately 100 days. Sequential administration of Ad-E4-122aT-AHAFIX resulted in more than 100% of the plasma hFIX activity levels for more than 100 days and rescued the bleeding phenotypes of hemophilia B mice. In addition, immunotolerance to hFIX was induced by Ad-E4-122aT-AHAFIX administration in neonatal hemophilia B mice. These results indicated that Ad-E4-122aT is a promising gene delivery vector for neonatal or infant gene therapy for hemophilia B.

Differential adenoassociated virus vector-driven expression of a neuropeptide Y gene in primary rat brain astroglial cultures after transfection with Sendai virosomes versus Lipofectin.

Science.gov (United States)

de Fiebre, C M; Wu, P; Notabartolo, D; Millard, W J; Meyer, E M

1994-06-01

The ability of Sendai virosomes or Lipofectin to introduce an AAV vector into primary rat brain astroglial cultures was characterized. The pJDT95npy vector was constructed by inserting rat NPY cDNA downstream from the indigenous AAV p5, p19 and p40 promoters in pJDT95. Lipofectin-mediated transfection with pJDT95npy (10 micrograms) resulted in pronounced expression of several NPY mRNA species: p5-driven (3.3 kb), p19-driven (2.7 kb) and p40-driven (0.6, 0.8, 1.1, and 1.8 kb). Exposure to virosomally encapsulated pJDT95npy (50 or 100 ng) resulted in transient expression of some p40-driven mRNA species (0.8 and 1.8 kb). Neither method produced astroglia cells which synthesized mature NPY immunoreactivity. This demonstrates that an AAV-derived vector can drive gene expression in astroglia, that Sendai virosomes can infuse vectors into astroglia, but that the amount of DNA infused in this manner may limit long term expression.

Emerging spatial patterns in Antarctic prokaryotes

Directory of Open Access Journals (Sweden)

Chun Wie eChong

2015-09-01

Full Text Available Recent advances in knowledge of patterns of biogeography in terrestrial eukaryotic organisms have led to a fundamental paradigm shift in understanding of the controls and history of life on land in Antarctica, and its interactions over the long term with the glaciological and geological processes that have shaped the continent. However, while it has long been recognized that the terrestrial ecosystems of Antarctica are dominated by microbes and their processes, knowledge of microbial diversity and distributions has lagged far behind that of the macroscopic eukaryote organisms. Increasing human contact with and activity in the continent is leading to risks of biological contamination and change in a region whose isolation has protected it for millions of years at least; these risks may be particularly acute for microbial communities which have, as yet, received scant recognition and attention. Even a matter apparently as straightforward as Protected Area designation in Antarctica requires robust biodiversity data which, in most parts of the continent, remain almost completely unavailable. A range of important contributing factors mean that it is now timely to reconsider the state of knowledge of Antarctic terrestrial prokaryotes. Rapid advances in molecular biological approaches are increasingly demonstrating that bacterial diversity in Antarctica may be far greater than previously thought, and that there is overlap in the environmental controls affecting both Antarctic prokaryotic and eukaryotic communities. Bacterial dispersal mechanisms and colonization patterns remain largely unaddressed, although evidence for regional evolutionary differentiation is rapidly accruing and, with this, there is increasing appreciation of patterns in regional bacterial biogeography in this large part of the globe. In this review, we set out to describe the state of knowledge of Antarctic prokaryote diversity patterns, drawing analogy with those of eukaryote

Proteolytic enzymes in seawater: contribution of prokaryotes and protists

Science.gov (United States)

Obayashi, Y.; Suzuki, S.

2016-02-01

Proteolytic enzyme is one of the major catalysts of microbial processing of organic matter in biogeochemical cycle. Here we summarize some of our studies about proteases in seawater, including 1) distribution of protease activities in coastal and oceanic seawater, 2) responses of microbial community and protease activities in seawater to organic matter amending, and 3) possible contribution of heterotrophic protists besides prokaryotes to proteases in seawater, to clarify cleared facts and remaining questions. Activities of aminopeptidases, trypsin-type and chymotrypsin-type proteases were detected from both coastal and oceanic seawater by using MCA-substrate assay. Significant activities were detected from not only particulate (cell-associated) fraction but also dissolved fraction of seawater, especially for trypsin-type and chymotrypsin-type proteases. Hydrolytic enzymes in seawater have been commonly thought to be mainly derived from heterotrophic prokaryotes; however, it was difficult to determine actual source organisms of dissolved enzymes in natural seawater. Our experiment with addition of dissolved protein to subtropical oligotrophic Pacific water showed drastically enhancement of the protease activities especially aminopeptidases in seawater, and the prokaryotic community structure simultaneously changed to be dominant of Bacteroidetes, indicating that heterotrophic bacteria were actually one of the sources of proteases in seawater. Another microcosm experiment with free-living marine heterotrophic ciliate Paranophrys marina together with an associated bacterium showed that extracellular trypsin-type activity was mainly attributed to the ciliate. The protist seemed to work in organic matter digestion in addition to be a grazer. From the results, we propose a system of organic matter digestion by prokaryotes and protists in aquatic environments, although their actual contribution in natural environments should be estimated in future studies.

Expression profile of genes during resistance reversal in a temephos selected strain of the dengue vector, Aedes aegypti.

Directory of Open Access Journals (Sweden)

Clare Strode

Full Text Available BACKGROUND: The mosquito Aedes aegypti is one of the most important disease vectors because it transmits two major arboviruses, dengue and yellow fever, which cause significant global morbidity and mortality. Chemical insecticides form the cornerstone of vector control. The organophosphate temephos a larvicide recommended by WHO for controlling Ae. aegypti, however, resistance to this compound has been reported in many countries, including Brazil. METHODOLOGY/PRINCIPAL FINDINGS: The aim of this study was to identify genes implicated in metabolic resistance in an Ae. aegypti temephos resistant strain, named RecR, through microarray analysis. We utilized a custom 'Ae. aegypti detox chip' and validated microarray data through RT-PCR comparing susceptible and resistant individuals. In addition, we analyzed gene expression in 4(th instar larvae from a reversed susceptible strain (RecRev, exposed and unexposed to temephos. The results obtained revealed a set of 13 and 6 genes significantly over expressed in resistant adult mosquitoes and larvae, respectively. One of these genes, the cytochrome P450 CYP6N12, was up-regulated in both stages. RT-PCR confirmed the microarray results and, additionally, showed no difference in gene expression between temephos exposed and unexposed RecRev mosquitoes. This suggested that the differences in the transcript profiles among the strains are heritable due to a selection process and are not caused by immediate insecticide exposure. Reversal of temephos resistance was demonstrated and, importantly, there was a positive correlation between a decrease in the resistance ratio and an accompanying decrease in the expression levels of previously over expressed genes. Some of the genes identified here have also been implicated in metabolic resistance in other mosquito species and insecticide resistant populations of Ae. aegypti. CONCLUSIONS/SIGNIFICANCE: The identification of gene expression signatures associated to

Metabolic profiles of prokaryotic and eukaryotic communities in deep-sea sponge Neamphius huxleyi [corrected]. indicated by metagenomics.

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Li, Zhi-Yong; Wang, Yue-Zhu; He, Li-Ming; Zheng, Hua-Jun

2014-01-27

The whole metabolism of a sponge holobiont and the respective contributions of prokaryotic and eukaryotic symbionts and their associations with the sponge host remain largely unclear. Meanwhile, compared with shallow water sponges, deep-sea sponges are rarely understood. Here we report the metagenomic exploration of deep-sea sponge Neamphius huxleyi [corrected] . at the whole community level. Metagenomic data showed phylogenetically diverse prokaryotes and eukaryotes in Neamphius huxleyi [corrected]. MEGAN and gene enrichment analyses indicated different metabolic potentials of prokaryotic symbionts from eukaryotic symbionts, especially in nitrogen and carbon metabolisms, and their molecular interactions with the sponge host. These results supported the hypothesis that prokaryotic and eukaryotic symbionts have different ecological roles and relationships with sponge host. Moreover, vigorous denitrification, and CO2 fixation by chemoautotrophic prokaryotes were suggested for this deep-sea sponge. The study provided novel insights into the respective potentials of prokaryotic and eukaryotic symbionts and their associations with deep-sea sponge Neamphius huxleyi [corrected].

Immunological characteristics of outer membrane protein omp31 of goat Brucella and its monoclonal antibody.

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Zheng, W Y; Wang, Y; Zhang, Z C; Yan, F

2015-10-05

We examined the immunological characteristics of outer membrane protein omp31 of goat Brucella and its monoclonal antibody. Genomic DNA from the M5 strain of goat Brucella was amplified by polymerase chain reaction and cloned into the prokaryotic expression vector pGEX-4T-1. The expression and immunological characteristics of the fusion protein GST-omp31 were subjected to preliminary western blot detection with goat Brucella rabbit immune serum. The Brucella immunized BALB/c mouse serum was detected using purified protein. The high-potency mouse splenocytes and myeloma Sp2/0 cells were fused. Positive clones were screened by enzyme-linked immunosorbent assay to establish a hybridoma cell line. Mice were inoculated intraperitoneally with hybridoma cells to prepare ascites. The mAb was purified using the n-caprylic acid-ammonium sulfate method. The characteristics of mAb were examined using western blotting and enzyme-linked immunosorbent assay. A 680-base pair band was observed after polymerase chain reaction. Enzyme digestion identification and sequencing showed that the pGEX-4T-1-omp31 prokaryotic expression vector was successfully established; a target band of approximately 57 kDa with an apparent molecular weight consistent with the size of the target fusion protein. At 25°C, the expression of soluble expression increased significantly; the fusion protein GST-omp31 was detected by western blotting. Anti-omp31 protein mAb was obtained from 2 strains of Brucella. The antibody showed strong specificity and sensitivity and did not cross-react with Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Mycobacterium tuberculosis, or Bacillus pyocyaneus. The pGEX-4T-1-omp31 prokaryotic expression vector was successfully established and showed good immunogenicity. The antibody also showed strong specificity and good sensitivity.

Preparation and characterization of the polyclonal antibody against ...

African Journals Online (AJOL)

To prepare the polyclonal antibody against GAR domain, cDNA encoding 466 amino acids protein of GAR domain was amplified from MG-63 cell by RT-PCR. The amplified cDNA, that exhibited 99% identity to the published sequence, was cloned into prokaryotic expression vector pQE-80L for the expression of GAR ...

Gene duplications in prokaryotes can be associated with environmental adaptation

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Lempicki Richard A

2010-10-01

Full Text Available Abstract Background Gene duplication is a normal evolutionary process. If there is no selective advantage in keeping the duplicated gene, it is usually reduced to a pseudogene and disappears from the genome. However, some paralogs are retained. These gene products are likely to be beneficial to the organism, e.g. in adaptation to new environmental conditions. The aim of our analysis is to investigate the properties of paralog-forming genes in prokaryotes, and to analyse the role of these retained paralogs by relating gene properties to life style of the corresponding prokaryotes. Results Paralogs were identified in a number of prokaryotes, and these paralogs were compared to singletons of persistent orthologs based on functional classification. This showed that the paralogs were associated with for example energy production, cell motility, ion transport, and defence mechanisms. A statistical overrepresentation analysis of gene and protein annotations was based on paralogs of the 200 prokaryotes with the highest fraction of paralog-forming genes. Biclustering of overrepresented gene ontology terms versus species was used to identify clusters of properties associated with clusters of species. The clusters were classified using similarity scores on properties and species to identify interesting clusters, and a subset of clusters were analysed by comparison to literature data. This analysis showed that paralogs often are associated with properties that are important for survival and proliferation of the specific organisms. This includes processes like ion transport, locomotion, chemotaxis and photosynthesis. However, the analysis also showed that the gene ontology terms sometimes were too general, imprecise or even misleading for automatic analysis. Conclusions Properties described by gene ontology terms identified in the overrepresentation analysis are often consistent with individual prokaryote lifestyles and are likely to give a competitive

Superior induction and maintenance of protective CD8 T cells in mice infected with mouse cytomegalovirus vector expressing RAE-1γ.

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Trsan, Tihana; Busche, Andreas; Abram, Maja; Wensveen, Felix M; Lemmermann, Niels A; Arapovic, Maja; Babic, Marina; Tomic, Adriana; Golemac, Mijo; Brinkmann, Melanie M; Jäger, Wiebke; Oxenius, Annette; Polic, Bojan; Krmpotic, Astrid; Messerle, Martin; Jonjic, Stipan

2013-10-08

Due to a unique pattern of CD8 T-cell response induced by cytomegaloviruses (CMVs), live attenuated CMVs are attractive candidates for vaccine vectors for a number of clinically relevant infections and tumors. NKG2D is one of the most important activating NK cell receptors that plays a role in costimulation of CD8 T cells. Here we demonstrate that the expression of CD8 T-cell epitope of Listeria monocytogenes by a recombinant mouse CMV (MCMV) expressing the NKG2D ligand retinoic acid early-inducible protein 1-gamma (RAE-1γ) dramatically enhanced the effectiveness and longevity of epitope-specific CD8 T-cell response and conferred protection against a subsequent challenge infection with Listeria monocytogenes. Unexpectedly, the attenuated growth in vivo of the CMV vector expressing RAE-1γ and its capacity to enhance specific CD8 T-cell response were preserved even in mice lacking NKG2D, implying additional immune function for RAE-1γ beyond engagement of NKG2D. Thus, vectors expressing RAE-1γ represent a promising approach in the development of CD8 T-cell-based vaccines.

A recombinant E1-deleted porcine adenovirus-3 as an expression vector

International Nuclear Information System (INIS)

Zakhartchouk, Alexander; Zhou Yan; Tikoo, Suresh Kumar

2003-01-01

Replication-defective E1-deleted porcine adenoviruses (PAVs) are attractive vectors for vaccination. As a prerequisite for generating PAV-3 vectors containing complete deletion of E1, we transfected VIDO R1 cells (fetal porcine retina cells transformed with E1 region of human adenovirus 5) with a construct containing PAV-3 E1B large coding sequences under the control of HCMV promoter. A cell line named VR1BL could be isolated that expressed E1B large of PAV-3 and also complemented PAV214 (E1A+E1B small deleted). The VR1BL cells could be efficiently transfected with DNA and allowed the rescue and propagation of recombinant PAV507 containing a triple stop codon inserted in the E1B large coding sequence. In addition, recombinant PAV227 containing complete deletion of E1 (E1A+E1B small + E1B large ) could be successfully rescued using VR1BL cell line. Recombinant PAV227 replicated as efficiently as wild-type in VR1BL cells but not in VIDO R1 cells, suggesting that E1B large was essential for replication of PAV-3. Next, we constructed recombinant PAV219 by inserting green fluorescent (GFP) protein gene flanked by a promoter and a poly(A) in the E1 region of the PAV227 genome. We demonstrated that PAV219 was able to transduce and direct expression of GFP in some human cell lines

A synthetic system for expression of components of a bacterial microcompartment.

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Sargent, Frank; Davidson, Fordyce A; Kelly, Ciarán L; Binny, Rachelle; Christodoulides, Natasha; Gibson, David; Johansson, Emelie; Kozyrska, Katarzyna; Lado, Lucia Licandro; Maccallum, Jane; Montague, Rachel; Ortmann, Brian; Owen, Richard; Coulthurst, Sarah J; Dupuy, Lionel; Prescott, Alan R; Palmer, Tracy

2013-11-01

In general, prokaryotes are considered to be single-celled organisms that lack internal membrane-bound organelles. However, many bacteria produce proteinaceous microcompartments that serve a similar purpose, i.e. to concentrate specific enzymic reactions together or to shield the wider cytoplasm from toxic metabolic intermediates. In this paper, a synthetic operon encoding the key structural components of a microcompartment was designed based on the genes for the Salmonella propanediol utilization (Pdu) microcompartment. The genes chosen included pduA, -B, -J, -K, -N, -T and -U, and each was shown to produce protein in an Escherichia coli chassis. In parallel, a set of compatible vectors designed to express non-native cargo proteins was also designed and tested. Engineered hexa-His tags allowed isolation of the components of the microcompartments together with co-expressed, untagged, cargo proteins. Finally, an in vivo protease accessibility assay suggested that a PduD-GFP fusion could be protected from proteolysis when co-expressed with the synthetic microcompartment operon. This work gives encouragement that it may be possible to harness the genes encoding a non-native microcompartment for future biotechnological applications.

Inhibition of HBV replication by delivering the dual-gene expression vector pHsa-miR16-siRNA in HepG2.2.15 cells.

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Wei, Wei; Wang, Su-Fei; Yu, Bing; Ni, Ming

2017-12-01

This study aimed to construct the dual-gene expression vector pHsa-miR16-siRNA which can express human miR-16 and HBV X siRNA, and examine its regulatory effect on HBV gene expression in the HepG2.2.15 cell line. The expression vectors siR-1583 and pHsa-miR16-siRNA were designed and constructed. HepG2.2.15 cells were transfected with the empty vector, siR-1583, pmiR-16 and pHsa-miR16-siRNA, respectively. ELISA was performed to measure the expression of HBsAg and HBeAg in the culture supernatant 48 and72 h post transfection. Fluorescence quantitative PCR was used to measure the HBV mRNA degradation efficiency and HBV DNA copy number. The results showed that the expression of HBV genes was significantly inhibited in HepG2.2.15 cells transfected with siR-1583, pmiR-16 and pHsa-miR16-siRNA, respectively, when compared with that in cells transfected with the empty vectors, with the inhibitory effect of pHsa-miR16-siRNA being the most significant. ELISA showed that the inhibitory rates of HBsAg and HBeAg in pHsa-miR16-siRNA transfected cells were correspondingly 87.3% and 85.0% at 48 h, and 88.6% and 86.5% at 72 h post transfection (PHBV mRNA decreased by 80.2% (t=-99.22, PHBV DNA by 92.8% (t=-73.06, PHBV DNA copy number by 89.8% (t=-47.13, PHBV more efficiently than a single-gene expression vector.

Design and Potential of Non-Integrating Lentiviral Vectors

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Aaron Shaw

2014-01-01

Full Text Available Lentiviral vectors have demonstrated promising results in clinical trials that target cells of the hematopoietic system. For these applications, they are the vectors of choice since they provide stable integration into cells that will undergo extensive expansion in vivo. Unfortunately, integration can have unintended consequences including dysregulated cell growth. Therefore, lentiviral vectors that do not integrate are predicted to have a safer profile compared to integrating vectors and should be considered for applications where transient expression is required or for sustained episomal expression such as in quiescent cells. In this review, the system for generating lentiviral vectors will be described and used to illustrate how alterations in the viral integrase or vector Long Terminal Repeats have been used to generate vectors that lack the ability to integrate. In addition to their safety advantages, these non-integrating lentiviral vectors can be used when persistent expression would have adverse consequences. Vectors are currently in development for use in vaccinations, cancer therapy, site-directed gene insertions, gene disruption strategies, and cell reprogramming. Preclinical work will be described that illustrates the potential of this unique vector system in human gene therapy.

Neuron-specific RNA interference using lentiviral vectors

DEFF Research Database (Denmark)

Nielsen, Troels Tolstrup; Marion, Ingrid van; Hasholt, Lis

2009-01-01

BACKGROUND: Viral vectors have been used in several different settings for the delivery of small hairpin (sh) RNAs. However, most vectors have utilized ubiquitously-expressing polymerase (pol) III promoters to drive expression of the hairpin as a result of the strict requirement for precise...... transcriptional initiation and termination. Recently, pol II promoters have been used to construct vectors for RNA interference (RNAi). By embedding the shRNA into a micro RNA-context (miRNA) the endogenous miRNA processing machinery is exploited to achieve the mature synthetic miRNA (smiRNA), thereby expanding...... the possible promoter choices and eventually allowing cell type specific down-regulation of target genes. METHODS: In the present study, we constructed lentiviral vectors expressing smiRNAs under the control of pol II promoters to knockdown gene expression in cell culture and in the brain. RESULTS: We...

Comparison of expression vectors in Lactobacillus reuteri strains.

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Lizier, Michela; Sarra, Pier G; Cauda, Roberto; Lucchini, Franco

2010-07-01

The synthesis of heterologous proteins in lactobacilli is strongly influenced by the promoter selected for the expression. In addition, the activity of the promoters themselves may vary among different bacterial hosts. Three different promoters were investigated for their capability to drive enhanced green fluorescent protein (EGFP) expression in Lactococcus lactis spp. cremoris MG1363, in Lactobacillus reuteri DSM 20016(T) and in five L. reuteri strains isolated from chicken crops. The promoters of the Lactobacillus acidophilus surface layer protein gene (slp), L. acidophilus lactate dehydrogenase gene (ldhL) and enterococcal rRNA adenine N-6-methyltransferase gene (ermB) were fused to the coding sequence of EGFP and inserted into the backbone of the pTRKH3 shuttle vector (pTRKH3-slpGFP, pTRKH3-ldhGFP, pTRKH3-ermGFP). Besides conventional analytical methods, a new quick fluorimetric approach was set up to quantify the EGFP fluorescence in transformed clones using the Qubit() fluorometer. ermB proved to be the most effective promoter in L. reuteri isolates, producing 3.90 x 10(-7) g of fluorescent EGFP (mL OD(stationary culture))(-1). Under the same conditions, the ldhL promoter produced 2.66 x 10(-7) g of fluorescent EGFP (mL OD(stationary culture))(-1). Even though the slp promoter was efficient in L. lactis spp. cremoris MG1363, it was nearly inactive both in L. reuteri DSM 20016(T) and in L. reuteri isolates.

Prokaryotic Expression and Serodiagnostic Potential of Glyceraldehyde-3-Phosphate Dehydrogenase and Thioredoxin Peroxidase from Baylisascaris schroederi

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Yu Li

2017-10-01

Full Text Available Baylisascaris schroederi, a roundworm parasite of giant pandas, badly affects the health of its hosts. Diagnosis of this disease currently depends mainly on sedimentation floatation and Polymerase Chain Reaction (PCR methods to detect the eggs. However, neither of these methods is suitable for diagnosis of early-stage panda baylisascariasis and no information on early diagnosis of this disease is available so far. Therefore, to develop an effective serologic diagnostic method, this study produced recombinant glyceraldehyde-3-phosphate dehydrogenase (GAPDH and thioredoxin peroxidase (Tpx proteins from B. schroederi using a prokaryotic expression system. We determined the immunological characteristics of these proteins and their location in the parasite. Indirect enzyme-linked immunosorbent assays (ELISAs were established to detect B. schroederi infection in giant pandas based on GAPDH and Tpx respectively. The open reading frame of the GAPDH gene (1083 bp encoded a 39 kDa protein, while the predicted molecular weight of Tpx (588 bp was 21.6 kDa. Western-blotting analysis revealed that both recombinant proteins could be recognized with positive serum of pandas infected with B. schroederi. Immunohistochemical staining showed that the endogenous GAPDH of B. schroederi was widely distributed in the worm while Tpx was mainly localized in the muscle, eggs, gut wall, uterus wall and hypodermis. Serological tests showed that the GAPDH-based indirect ELISA had a sensitivity of 95.83% and specificity of 100%, while the test using Tpx as the antigen had sensitivity of 75% and specificity of 91.7%. Thus, B. schroederi Tpx is unsuitable as a diagnostic antigen for baylisascariasis, but B. schroederi GAPDH is a good candidate diagnostic antigen for B. schroederi in pandas.

Prokaryotic photosynthesis and phototrophy illuminated

DEFF Research Database (Denmark)

Bryant, Donald A; Frigaard, Niels-Ulrik

2006-01-01

Genome sequencing projects are revealing new information about the distribution and evolution of photosynthesis and phototrophy. Although coverage of the five phyla containing photosynthetic prokaryotes (Chlorobi, Chloroflexi, Cyanobacteria, Proteobacteria and Firmicutes) is limited and uneven...... components that have not yet been described. Metagenomics has already shown how the relatively simple phototrophy based upon rhodopsins has spread laterally throughout Archaea, Bacteria and eukaryotes. In this review, we present examples that reflect recent advances in phototroph biology as a result...

GenoCAD Plant Grammar to Design Plant Expression Vectors for Promoter Analysis.

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Coll, Anna; Wilson, Mandy L; Gruden, Kristina; Peccoud, Jean

2016-01-01

With the rapid advances in prediction tools for discovery of new promoters and their cis-elements, there is a need to improve plant expression methodologies in order to facilitate a high-throughput functional validation of these promoters in planta. The promoter-reporter analysis is an indispensible approach for characterization of plant promoters. It requires the design of complex plant expression vectors, which can be challenging. Here, we describe the use of a plant grammar implemented in GenoCAD that will allow the users to quickly design constructs for promoter analysis experiments but also for other in planta functional studies. The GenoCAD plant grammar includes a library of plant biological parts organized in structural categories to facilitate their use and management and a set of rules that guides the process of assembling these biological parts into large constructs.

Efficient and sustained IGF-1 expression in the adipose tissue-derived stem cells mediated via a lentiviral vector.

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Chen, Ting; Huang, Dangsheng; Chen, Guanghui; Yang, Tingshu; Yi, Jun; Tian, Miao

2015-02-01

The adipose tissue-derived stem cells (ADSCs) represent a significant area of the cell therapy. Genetic modification of ADSCs may further improve their therapeutic potential. Here, we aimed to generate a lentiviral vector expressing insulin-like growth factor-I (IGF-1) and investigate the impact of IGF-1 transduction on the properties of cultured ADSCs. Isolated rat ADSCs were assessed by flow cytometric analysis. IGF-1 was cloned and inserted into the pLenO-DCE plasmid to acquire pLenO-DCE-IGF-1 plasmid. Lentivirus was enveloped with pRsv-REV, pMDlg-pRRE and pMD2G plasmids in 293T cells. The ADSCs were transfected with the vectors. And then IGF-1-induced anti-apoptosis was evaluated by annexin V-FITC. Besides, proliferation of cells was detected by MTT assay and EdU. Moreover, Akt phosphorylation was evaluated by Western blotting analysis. Stable expression of IGF-1 in ADSCs was confirmed. ADSCs were positive for CD90 and CD29, but negative for CD31, CD34 and CD45. The transduction of IGF-1 to the ADSCs caused a dramatic increase in P-Akt expression. Over-expression of IGF-1 in ADSCs could improve the paracine of IGF-1 in a time-dependent manner, but could not promote the proliferation of ADSCs. This study indicated that lentiviral vectors offered a promising mean of delivering IGF-1 to the ADSCs. Lentiviral-mediated over-expression of therapeutic IGF-1 gene in ADSCs could prolong the anti-apoptosis effect of IGF-1, which might be induced by the activation of the PI3K/Akt pathway. And our data would improve the efficacy of ADSC-based therapies.

Thiol biochemistry of prokaryotes

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Fahey, Robert C.

1986-01-01

The present studies have shown that GSH metabolism arose in the purple bacteria and cyanobacteria where it functions to protect against oxygen toxicity. Evidence was obtained indicating that GSH metabolism was incorporated into eucaryotes via the endosymbiosis giving rise to mitochrondria and chloroplasts. Aerobic bacteria lacking GSH utilize other thiols for apparently similar functions, the thiol being coenzyme A in Gram positive bacteria and chi-glutamylcysteine in the halobacteria. The thiol biochemistry of prokaryotes is thus seen to be much more highly diversified than that of eucaryotes and much remains to be learned about this subject.

A replicating plasmid-based vector for GFP expression in Mycoplasma hyopneumoniae.

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Ishag, H Z A; Liu, M J; Yang, R S; Xiong, Q Y; Feng, Z X; Shao, G Q

2016-04-28

Mycoplasma hyopneumoniae (M. hyopneumoniae) causes porcine enzootic pneumonia (PEP) that significantly affects the pig industry worldwide. Despite the availability of the whole genome sequence, studies on the pathogenesis of this organism have been limited due to the lack of a genetic manipulation system. Therefore, the aim of the current study was to generate a general GFP reporter vector based on a replicating plasmid. Here, we describe the feasibility of GFP reporter expression in M. hyopneumoniae (strain 168L) controlled by the p97 gene promoter of this mycoplasma. An expression plasmid (pMD18-TOgfp) containing the p97 gene promoter, and origin of replication (oriC) of M. hyopneumoniae, tetracycline resistant marker (tetM), and GFP was constructed and used to transform competent M. hyopneumoniae cells. We observed green fluorescence in M. hyopneumoniae transformants under fluorescence microscopy, which indicates that there was expression of the GFP reporter that was driven by the p97 gene promoter. Additionally, an electroporation method for M. hyopneumoniae with an efficiency of approximately 1 x 10(-6) transformants/μg plasmid DNA was optimized and is described herein. In conclusion, our data demonstrate the susceptibility of M. hyopneumoniae to genetic manipulation whereby foreign genes are expressed. This work may encourage the development of genetic tools to manipulate the genome of M. hyopneumoniae for functional genomic analyses.

The Influence of SV40 polyA on Gene Expression of Baculovirus Expression Vector Systems.

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Tamer Z Salem

Full Text Available The simian virus 40 polyadenylation signal (SV40 polyA has been routinely inserted downstream of the polyhedrin promoter in many baculovirus expression vector systems (BEVS. In the baculovirus prototype Autographa californica multiple nucleopolyhedrovirus (AcMNPV, the polyhedrin promoter (very late promoter transcribes its gene by a viral RNA polymerase therefore there is no supporting evidence that SV40 polyA is required for the proper gene expression under the polyhedrin promoter. Moreover, the effect of the SV40 polyA sequence on the polyhedrin promoter activity has not been tested either at its natural polyhedrin locus or in other loci in the viral genome. In order to test the significance of adding the SV40 polyA sequence on gene expression, the expression of the enhanced green fluorescent protein (egfp was evaluated with and without the presence of SV40 polyA under the control of the polyhedrin promoter at different genomic loci (polyherin, ecdysteroid UDP-glucosyltransferase (egt, and gp37. In this study, spectrofluorometry and western blot showed reduction of EGFP protein for all recombinant viruses with SV40 polyA, whereas qPCR showed an increase in the egfp mRNA levels. Therefore, we conclude that SV40 polyA increases mRNA levels but decreases protein production in the BEVS when the polyhedrin promoter is used at different loci. This work suggests that SV40 polyA in BEVSs should be replaced by an AcMNPV late gene polyA for optimal protein production or left untouched for optimal RNA production (RNA interference applications.

The Influence of SV40 polyA on Gene Expression of Baculovirus Expression Vector Systems

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Salem, Tamer Z.; Seaborn, Craig P.; Turney, Colin M.; Xue, Jianli; Shang, Hui; Cheng, Xiao-Wen

2015-01-01

The simian virus 40 polyadenylation signal (SV40 polyA) has been routinely inserted downstream of the polyhedrin promoter in many baculovirus expression vector systems (BEVS). In the baculovirus prototype Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the polyhedrin promoter (very late promoter) transcribes its gene by a viral RNA polymerase therefore there is no supporting evidence that SV40 polyA is required for the proper gene expression under the polyhedrin promoter. Moreover, the effect of the SV40 polyA sequence on the polyhedrin promoter activity has not been tested either at its natural polyhedrin locus or in other loci in the viral genome. In order to test the significance of adding the SV40 polyA sequence on gene expression, the expression of the enhanced green fluorescent protein (egfp) was evaluated with and without the presence of SV40 polyA under the control of the polyhedrin promoter at different genomic loci (polyherin, ecdysteroid UDP-glucosyltransferase (egt), and gp37). In this study, spectrofluorometry and western blot showed reduction of EGFP protein for all recombinant viruses with SV40 polyA, whereas qPCR showed an increase in the egfp mRNA levels. Therefore, we conclude that SV40 polyA increases mRNA levels but decreases protein production in the BEVS when the polyhedrin promoter is used at different loci. This work suggests that SV40 polyA in BEVSs should be replaced by an AcMNPV late gene polyA for optimal protein production or left untouched for optimal RNA production (RNA interference applications). PMID:26659470

[Expression and activity determination of recombinant capsid protein VP2 gene of enterovirus type 71].

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Huang, Xueyong; Liu, Guohua; Hu, Xiaoning; Du, Yanhua; Li, Xingle; Xu, Yuling; Chen, Haomin; Xu, Bianli

2014-04-01

To clone and express the recombinant capsid protein VP2 of enterovirus type 71 (EV71) and to identify the immune activity of expressed protein in order to build a basis for the investigation work of vaccine and diagnostic antigen. VP2 gene of EV71 was amplified by PCR, and then was cut by restriction enzyme and inserted into expression vector pMAL-c2X. The positive recombinants were transferred into E.coli TB1, the genetically engineered bacteria including pMAL-c2X-VP2 plasmids were induced by isopropyl thiogalactoside ( IPTG) , and the expression products were analyzed by SDS-PAGE and western blotting method. EV71 IgM antibody detection method by ELISA was set up, and the sensitivity and specificity of this method was assessed; 60 neutralizing antibody positive serum samples from hand foot and mouth disease (HFMD) patients were determined, of which 52 samples were positive and 8 samples were negative; a total of 88 acute phase serum samples of HFMD patients diagnosed in clinical were also detected. VP2 gene of 762 bp was obtained by PCR, the gene segment inserted into the recombinant vector was identified using restriction enzyme digestion. The recombinant vector could express a specific about 71 500 fusion protein in E.coli by SDS-PAGE. The purified recombinant protein of EV71-VP2 can react with the serum of HFMD patients to produce a specific band by western blotting. The sensitivity and specificity of ELISA was 87% and 83%, respectively. Of the 88 acute phase serum samples from children with HFMD, 48 samples (55%) were positive by the ELISA assay. VP2 gene of EV71 has been cloned and a prokaryotic high expression system for VP2 gene was successfully constructed in the present study. The recombination EV71-VP2 has well antigenicity, which could be useful for developing diagnose reagent or vaccine of EV71.

A computational genomics pipeline for prokaryotic sequencing projects.

Science.gov (United States)

Kislyuk, Andrey O; Katz, Lee S; Agrawal, Sonia; Hagen, Matthew S; Conley, Andrew B; Jayaraman, Pushkala; Nelakuditi, Viswateja; Humphrey, Jay C; Sammons, Scott A; Govil, Dhwani; Mair, Raydel D; Tatti, Kathleen M; Tondella, Maria L; Harcourt, Brian H; Mayer, Leonard W; Jordan, I King

2010-08-01

New sequencing technologies have accelerated research on prokaryotic genomes and have made genome sequencing operations outside major genome sequencing centers routine. However, no off-the-shelf solution exists for the combined assembly, gene prediction, genome annotation and data presentation necessary to interpret sequencing data. The resulting requirement to invest significant resources into custom informatics support for genome sequencing projects remains a major impediment to the accessibility of high-throughput sequence data. We present a self-contained, automated high-throughput open source genome sequencing and computational genomics pipeline suitable for prokaryotic sequencing projects. The pipeline has been used at the Georgia Institute of Technology and the Centers for Disease Control and Prevention for the analysis of Neisseria meningitidis and Bordetella bronchiseptica genomes. The pipeline is capable of enhanced or manually assisted reference-based assembly using multiple assemblers and modes; gene predictor combining; and functional annotation of genes and gene products. Because every component of the pipeline is executed on a local machine with no need to access resources over the Internet, the pipeline is suitable for projects of a sensitive nature. Annotation of virulence-related features makes the pipeline particularly useful for projects working with pathogenic prokaryotes. The pipeline is licensed under the open-source GNU General Public License and available at the Georgia Tech Neisseria Base (http://nbase.biology.gatech.edu/). The pipeline is implemented with a combination of Perl, Bourne Shell and MySQL and is compatible with Linux and other Unix systems.

Support vector machine-based facial-expression recognition method combining shape and appearance

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Han, Eun Jung; Kang, Byung Jun; Park, Kang Ryoung; Lee, Sangyoun

2010-11-01

Facial expression recognition can be widely used for various applications, such as emotion-based human-machine interaction, intelligent robot interfaces, face recognition robust to expression variation, etc. Previous studies have been classified as either shape- or appearance-based recognition. The shape-based method has the disadvantage that the individual variance of facial feature points exists irrespective of similar expressions, which can cause a reduction of the recognition accuracy. The appearance-based method has a limitation in that the textural information of the face is very sensitive to variations in illumination. To overcome these problems, a new facial-expression recognition method is proposed, which combines both shape and appearance information, based on the support vector machine (SVM). This research is novel in the following three ways as compared to previous works. First, the facial feature points are automatically detected by using an active appearance model. From these, the shape-based recognition is performed by using the ratios between the facial feature points based on the facial-action coding system. Second, the SVM, which is trained to recognize the same and different expression classes, is proposed to combine two matching scores obtained from the shape- and appearance-based recognitions. Finally, a single SVM is trained to discriminate four different expressions, such as neutral, a smile, anger, and a scream. By determining the expression of the input facial image whose SVM output is at a minimum, the accuracy of the expression recognition is much enhanced. The experimental results showed that the recognition accuracy of the proposed method was better than previous researches and other fusion methods.

Vector 33: A reduce program for vector algebra and calculus in orthogonal curvilinear coordinates

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Harper, David

1989-06-01

This paper describes a package with enables REDUCE 3.3 to perform algebra and calculus operations upon vectors. Basic algebraic operations between vectors and between scalars and vectors are provided, including scalar (dot) product and vector (cross) product. The vector differential operators curl, divergence, gradient and Laplacian are also defined, and are valid in any orthogonal curvilinear coordinate system. The package is written in RLISP to allow algebra and calculus to be performed using notation identical to that for operations. Scalars and vectors can be mixed quite freely in the same expression. The package will be of interest to mathematicians, engineers and scientists who need to perform vector calculations in orthogonal curvilinear coordinates.

Advanced technologies for improved expression of recombinant proteins in bacteria: perspectives and applications.

Science.gov (United States)

Gupta, Sanjeev K; Shukla, Pratyoosh

2016-12-01

Prokaryotic expression systems are superior in producing valuable recombinant proteins, enzymes and therapeutic products. Conventional microbial technology is evolving gradually and amalgamated with advanced technologies in order to give rise to improved processes for the production of metabolites, recombinant biopharmaceuticals and industrial enzymes. Recently, several novel approaches have been employed in a bacterial expression platform to improve recombinant protein expression. These approaches involve metabolic engineering, use of strong promoters, novel vector elements such as inducers and enhancers, protein tags, secretion signals, high-throughput devices for cloning and process screening as well as fermentation technologies. Advancement of the novel technologies in E. coli systems led to the production of "difficult to express" complex products including small peptides, antibody fragments, few proteins and full-length aglycosylated monoclonal antibodies in considerable large quantity. Wacker's secretion technologies, Pfenex system, inducers, cell-free systems, strain engineering for post-translational modification, such as disulfide bridging and bacterial N-glycosylation, are still under evaluation for the production of complex proteins and peptides in E. coli in an efficient manner. This appraisal provides an impression of expression technologies developed in recent times for enhanced production of heterologous proteins in E. coli which are of foremost importance for diverse applications in microbiology and biopharmaceutical production.

Expression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori

Science.gov (United States)

Hasanzadeh, Leila; Ghaznavi-Rad, Ehsanollah; Soufian, Safieh; Farjadi, Vahideh; Abtahi, Hamid

2013-01-01

Objective(s) : Helicobacter pylori, a human specific gastric pathogen is a causative agent of chronic active gastritis. The vacuolating cytotoxin (VacA) is an effective virulence factor involved in gastric injury. The aim of this study was to construct a recombinant protein containing antigenic region of VacA gene and determine its antigenicity. Materials and Methods: The antigenic region of VacA gene was detected by bioinformatics methods. The polymerase chain reaction method was used to amplify a highly antigenic region of VacA gene from chromosomal DNA of H. pylori. The eluted product was cloned into the prokaryotic expression vector pET32a. The target protein was expressed in the Escherichia coli BL21 (DE3) pLysS. The bacteria including pET32a-VacA plasmids were induced by IPTG. The antigenicity was finally studied by western blotting using sera of 15 H. pylori infected patients after purification. Results: Enzyme digestion analysis, PCR and DNA sequencing results showed that the target gene was inserted correctly into the recombinant vector. The expressed protein was purified successfully via affinity chromatography. Data indicated that antigenic region of VacA protein from Helicobacter pylori was recognized by all 15 patient’s sera. Conclusion : Our data showed that antigenic region of VacA protein can be expressed by in E. co.li. This protein was recognized by sera patients suffering from H. pylori infection. the recombinant protein has similar epitopes and close antigenic properties to the natural form of this antigen. Recombinant antigenic region of VacA protein also seems to be a promising antigen for protective and serologic diagnosis . PMID:23997913

Expression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori

Directory of Open Access Journals (Sweden)

Leila Hasanzadeh

2013-07-01

Full Text Available Objective(s: Helicobacter pylori, a human specific gastric pathogen is a causative agent of chronic active gastritis. The vacuolating cytotoxin (VacA is an effective virulence factor involved in gastric injury. The aim of this study was to construct a recombinant protein containing antigenic region of VacA gene and determine its antigenicity.   Materials and Methods: The antigenic region of VacA gene was detected by bioinformatics methods. The polymerase chain reaction method was used to amplify a highly antigenic region of VacA gene from chromosomal DNA of H. pylori. The eluted product was cloned into the prokaryotic expression vector pET32a. The target protein was expressed in the Escherichia coli BL21 (DE3 pLysS. The bacteria including pET32a-VacA plasmids were induced by IPTG. The antigenicity was finally studied by western blotting using sera of 15 H. pylori infected patients after purification. Results: Enzyme digestion analysis, PCR and DNA sequencing results showed that the target gene was inserted correctly into the recombinant vector. The expressed protein was purified successfully via affinity chromatography. Data indicated that antigenic region of VacA protein from Helicobacter pylori was recognized by all 15 patient’s sera. Conclusion : Our data showed that antigenic region of VacA protein can be expressed by in E. co.li. This protein was recognized by sera patients suffering from H. pylori infection. the recombinant protein has similar epitopes and close antigenic properties to the natural form of this antigen. Recombinant antigenic region of VacA protein also seems to be a promising antigen for protective and serologic diagnosis .

High-titer recombinant adeno-associated virus production utilizing a recombinant herpes simplex virus type I vector expressing AAV-2 Rep and Cap.

Science.gov (United States)

Conway, J E; Rhys, C M; Zolotukhin, I; Zolotukhin, S; Muzyczka, N; Hayward, G S; Byrne, B J

1999-06-01

Recombinant adeno-associated virus type 2 (rAAV) vectors have recently been used to achieve long-term, high level transduction in vivo. Further development of rAAV vectors for clinical use requires significant technological improvements in large-scale vector production. In order to facilitate the production of rAAV vectors, a recombinant herpes simplex virus type I vector (rHSV-1) which does not produce ICP27, has been engineered to express the AAV-2 rep and cap genes. The optimal dose of this vector, d27.1-rc, for AAV production has been determined and results in a yield of 380 expression units (EU) of AAV-GFP produced from 293 cells following transfection with AAV-GFP plasmid DNA. In addition, d27.1-rc was also efficient at producing rAAV from cell lines that have an integrated AAV-GFP provirus. Up to 480 EU/cell of AAV-GFP could be produced from the cell line GFP-92, a proviral, 293 derived cell line. Effective amplification of rAAV vectors introduced into 293 cells by infection was also demonstrated. Passage of rAAV with d27. 1-rc results in up to 200-fold amplification of AAV-GFP with each passage after coinfection of the vectors. Efficient, large-scale production (>109 cells) of AAV-GFP from a proviral cell line was also achieved and these stocks were free of replication-competent AAV. The described rHSV-1 vector provides a novel, simple and flexible way to introduce the AAV-2 rep and cap genes and helper virus functions required to produce high-titer rAAV preparations from any rAAV proviral construct. The efficiency and potential for scalable delivery of d27.1-rc to producer cell cultures should facilitate the production of sufficient quantities of rAAV vectors for clinical application.

An easy method for preparation of Cre-loxP regulated fluorescent adenoviral expression vectors and its application for direct reprogramming into hepatocytes

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Chitose Kurihara

2016-12-01

Full Text Available The recombinant adenoviral gene expression system is a powerful tool for gene delivery. However, it is difficult to obtain high titers of infectious virus, principally due to the toxicity of the expressed gene which affects on virus replication in the host HEK293 cells. To avoid these problems, we generated a Cre-loxP-regulated fluorescent universal vector (termed pAxCALRL. This vector produces recombinant adenoviruses that express the red fluorescent protein (RFP instead of the inserted gene during proliferation, which limits toxicity and can be used to monitor viral replication. Expression of the gene of interest is induced by co-infection with an adenovirus that expresses Cre-recombinase (AxCANCre. Recombinant adenovirus produced by this system that express Hnf4α and Foxa2 were used to reprogram mouse embryo fibroblast (MEF into induced-hepatocyte-like cells (iHep following several rounds of infection, demonstrating the efficacy of this new system.

Effects of sodium azide on the abundance of prokaryotes and viruses in marine samples.

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Christian Winter

Full Text Available Flow cytometry is set to become the standard method for enumerating prokaryotes and viruses in marine samples. However, the samples need to be flash-frozen in liquid nitrogen directly after aldehyde fixation. Because liquid nitrogen may not always be available, we tested the potential of sodium azide as a preservative for prokaryotes and viruses in marine samples as a possible alternative. For that we conducted incubation experiments with untreated and sodium azide treated marine water samples at 4°C and room temperature. The data indicate that sodium azide cannot be used to maintain marine samples used for the enumeration of prokaryotes and viruses.

Genetic manipulation of endosymbionts to control vector and vector borne diseases

Directory of Open Access Journals (Sweden)

Jay Prakash Gupta

Full Text Available Vector borne diseases (VBD are on the rise because of failure of the existing methods of control of vector and vector borne diseases and the climate change. A steep rise of VBDs are due to several factors like selection of insecticide resistant vector population, drug resistant parasite population and lack of effective vaccines against the VBDs. Environmental pollution, public health hazard and insecticide resistant vector population indicate that the insecticides are no longer a sustainable control method of vector and vector-borne diseases. Amongst the various alternative control strategies, symbiont based approach utilizing endosymbionts of arthropod vectors could be explored to control the vector and vector borne diseases. The endosymbiont population of arthropod vectors could be exploited in different ways viz., as a chemotherapeutic target, vaccine target for the control of vectors. Expression of molecules with antiparasitic activity by genetically transformed symbiotic bacteria of disease-transmitting arthropods may serve as a powerful approach to control certain arthropod-borne diseases. Genetic transformation of symbiotic bacteria of the arthropod vector to alter the vector’s ability to transmit pathogen is an alternative means of blocking the transmission of VBDs. In Indian scenario, where dengue, chikungunya, malaria and filariosis are prevalent, paratransgenic based approach can be used effectively. [Vet World 2012; 5(9.000: 571-576

[Experimental interaction of halophilic prokaryotes and opportunistic bacteria in brine].

Science.gov (United States)

Selivanova, E A; Nemtseva, N V

2013-01-01

Study the effect of extremely halophilic archaea and moderately halophilic bacteria on preservation of opportunistic bacteria in brine. 17 strains of moderately halophilic bacteria and 2 strains of extremely halophilic archaea were isolated from continental hypersaline lake Razval of Sol-Iletsk area of Orenburg Region. Identification of pure cultures of prokaryotes was carried out taking into account their phenotype properties and based on determination of 16S RNA gene sequence. The effect of halophilic prokaryote on elimination of Escherichia coli from brine was evaluated during co-cultivation. Antagonistic activity of cell extracts of the studied microorganisms was evaluated by photometric method. A more prolonged preservation of an E. coli strain in brine in the presence of live cells of extremely halophilic archaea Halorubrum tebenquichense and moderately halophilic bacteria Marinococcus halophilus was established. Extracts of cells of extremely halophilic archaea and moderately halophilic bacteria on the contrary displayed antagonistic activity. The protective effect of live cells of halophilic prokaryotes and antagonistic activity of their cell extracts change the period of conservation of opportunistic bacteria in brine that regulates inter-microbial interactions and changes the period of self-purification that reflects the sanitary condition of a hypersaline water body.

Depth Dependent Relationships between Temperature and Ocean Heterotrophic Prokaryotic Production

KAUST Repository

Lønborg, Christian

2016-06-07

Marine prokaryotes play a key role in cycling of organic matter and nutrients in the ocean. Using a unique dataset (>14,500 samples), we applied a space-for-time substitution analysis to assess the temperature dependence of prokaryotic heterotrophic production (PHP) in epi- (0-200 m), meso- (201-1000 m) and bathypelagic waters (1001-4000 m) of the global ocean. Here, we show that the temperature dependence of PHP is fundamentally different between these major oceanic depth layers, with an estimated ecosystem-level activation energy (E) of 36 ± 7 kJ mol for the epipelagic, 72 ± 15 kJ mol for the mesopelagic and 274 ± 65 kJ mol for the bathypelagic realm. We suggest that the increasing temperature dependence with depth is related to the parallel vertical gradient in the proportion of recalcitrant organic compounds. These Ea predict an increased PHP of about 5, 12, and 55% in the epi-, meso-, and bathypelagic ocean, respectively, in response to a water temperature increase by 1°C. Hence, there is indication that a major thus far underestimated feedback mechanism exists between future bathypelagic ocean warming and heterotrophic prokaryotic activity.

Depth Dependent Relationships between Temperature and Ocean Heterotrophic Prokaryotic Production

KAUST Repository

Lø nborg, Christian; Cuevas, L. Antonio; Reinthaler, Thomas; Herndl, Gerhard J.; Gasol, Josep M.; Moran, Xose Anxelu G.; Bates, Nicholas R.; á lvarez-Salgado, Xosé A.

2016-01-01

Marine prokaryotes play a key role in cycling of organic matter and nutrients in the ocean. Using a unique dataset (>14,500 samples), we applied a space-for-time substitution analysis to assess the temperature dependence of prokaryotic heterotrophic production (PHP) in epi- (0-200 m), meso- (201-1000 m) and bathypelagic waters (1001-4000 m) of the global ocean. Here, we show that the temperature dependence of PHP is fundamentally different between these major oceanic depth layers, with an estimated ecosystem-level activation energy (E) of 36 ± 7 kJ mol for the epipelagic, 72 ± 15 kJ mol for the mesopelagic and 274 ± 65 kJ mol for the bathypelagic realm. We suggest that the increasing temperature dependence with depth is related to the parallel vertical gradient in the proportion of recalcitrant organic compounds. These Ea predict an increased PHP of about 5, 12, and 55% in the epi-, meso-, and bathypelagic ocean, respectively, in response to a water temperature increase by 1°C. Hence, there is indication that a major thus far underestimated feedback mechanism exists between future bathypelagic ocean warming and heterotrophic prokaryotic activity.

Adeno-associated viral vector serotypes 1 and 5 targeted to the neonatal rat and pig striatum induce widespread transgene expression in the forebrain

DEFF Research Database (Denmark)

Kornum, Birgitte R; Stott, Simon R W; Mattsson, Bengt

2010-01-01

Viral vector-mediated gene transfer has emerged as a powerful means to target transgene expression in the central nervous system. Here we characterized the efficacy of serotypes 1 and 5 recombinant adeno-associated virus (rAAV) vectors encoding green fluorescent protein (GFP) after stereotaxic...

Expression of chicken parvovirus VP2 in chicken embryo fibroblasts requires codon optimization for production of naked DNA and vectored meleagrid herpesvirus type 1 vaccines.

Science.gov (United States)

Spatz, Stephen J; Volkening, Jeremy D; Mullis, Robert; Li, Fenglan; Mercado, John; Zsak, Laszlo

2013-10-01

Meleagrid herpesvirus type 1 (MeHV-1) is an ideal vector for the expression of antigens from pathogenic avian organisms in order to generate vaccines. Chicken parvovirus (ChPV) is a widespread infectious virus that causes serious disease in chickens. It is one of the etiological agents largely suspected in causing Runting Stunting Syndrome (RSS) in chickens. Initial attempts to express the wild-type gene encoding the capsid protein VP2 of ChPV by insertion into the thymidine kinase gene of MeHV-1 were unsuccessful. However, transient expression of a codon-optimized synthetic VP2 gene cloned into the bicistronic vector pIRES2-Ds-Red2, could be demonstrated by immunocytochemical staining of transfected chicken embryo fibroblasts (CEFs). Red fluorescence could also be detected in these transfected cells since the red fluorescent protein gene is downstream from the internal ribosome entry site (IRES). Strikingly, fluorescence could not be demonstrated in cells transiently transfected with the bicistronic vector containing the wild-type or non-codon-optimized VP2 gene. Immunocytochemical staining of these cells also failed to demonstrate expression of wild-type VP2, indicating that the lack of expression was at the RNA level and the VP2 protein was not toxic to CEFs. Chickens vaccinated with a DNA vaccine consisting of the bicistronic vector containing the codon-optimized VP2 elicited a humoral immune response as measured by a VP2-specific ELISA. This VP2 codon-optimized bicistronic cassette was rescued into the MeHV-1 genome generating a vectored vaccine against ChPV disease.

[Rapid expression and preparation of the recombinant fusion protein sTNFRII-gAD by adenovirus vector system].

Science.gov (United States)

Lu, Yue; Liu, Dan; Zhang, Xiaoren; Liu, Xuerong; Shen, Wei; Zheng, Gang; Liu, Yunfan; Dong, Xiaoyan; Wu, Xiaobing; Gao, Jimin

2011-08-01

We expressed and prepared the recombinant fusion protein sTNFRII-gAD consisted of soluble TNF receptor II and the globular domain of adiponectin by Adenovirus Vector System in mammalian BHK21c022 cells. First we used the adenovirus vector containing EGFP gene (rAd5-EGFP) to infect BHK21c022 cells at different MOI (from 0 to 1 000), and then evaluated their transduction efficiency and cytotoxicity. Similarly, we constructed the replication-deficient adenovirus type 5-sTNFRII-gAD (rAd5-sTNFRII-gAD). We collected the supernatants for Western blotting to determine the optimal MOI by comparing the expression levels of sTNFRII-gAD fusion protein, 48 h after the BHK21c022 cells were infected by rAd5-sTNFRII-gAD at different MOIs (from 0 to 1 000). Then, we chose rAd5-sTNFRII-gAD at MOI 100 to infect five bottles of BHK21c022 cells in 100 mL of serum-free chemically defined media 100 mL, harvested the supernatant every 48 h for 6 times, and condense and purify sTNFRII-gAD fusion protein by ammonium sulfate salt-out and size-exclusion chromatography, respectively. Finally, we analyzed anti-TNFalpha activity of sTNFRII-gAD fusion protein on L929 cells in vitro. The results showed that the number of BHK21c022 cells expressing EGFP protein was increased significantly with the increase of MOI. However, some cells died at MOI of 1 000 while there was no significant cytotoxicity at MOI from 0 to 100. Western blotting analysis showed that the more adenoviruses, the higher expression of sTNFRII-gAD fusion protein in the supernatant with the highest expression at MOI 1 000. We successfully obtained about 11 mg bioactive and purified sTNFRII-gAD fusion protein at last. The in vitro assay demonstrated that the sTNFRII-gAD fusion protein was potent to antagonize TNFalpha's cytotoxicity to L929 cells. Put together, we established a recombinant adenovirus vector/BHK21 cell expression system, characteristic of the efficient serum-free culture and easy scaling-up.

ConSpeciFix: Classifying prokaryotic species based on gene flow.

Science.gov (United States)

Bobay, Louis-Marie; Ellis, Brian Shin-Hua; Ochman, Howard

2018-05-16

Classification of prokaryotic species is usually based on sequence similarity thresholds, which are easy to apply but lack a biologically-relevant foundation. Here, we present ConSpeciFix, a program that classifies prokaryotes into species using criteria set forth by the Biological Species Concept, thereby unifying species definition in all domains of life. ConSpeciFix's webserver is freely available at www.conspecifix.com. The local version of the program can be freely downloaded from https://github.com/Bobay-Ochman/ConSpeciFix. ConSpeciFix is written in Python 2.7 and requires the following dependencies: Usearch, MCL, MAFFT and RAxML. ljbobay@uncg.edu.

Macro and Microelements Drive Diversity and Composition of Prokaryotic and Fungal Communities in Hypersaline Sediments and Saline-Alkaline Soils.

Science.gov (United States)

Liu, Kaihui; Ding, Xiaowei; Tang, Xiaofei; Wang, Jianjun; Li, Wenjun; Yan, Qingyun; Liu, Zhenghua

2018-01-01

Understanding the effects of environmental factors on microbial communities is critical for microbial ecology, but it remains challenging. In this study, we examined the diversity (alpha diversity) and community compositions (beta diversity) of prokaryotes and fungi in hypersaline sediments and salinized soils from northern China. Environmental variables were highly correlated, but they differed significantly between the sediments and saline soils. The compositions of prokaryotic and fungal communities in the hypersaline sediments were different from those in adjacent saline-alkaline soils, indicating a habitat-specific microbial distribution pattern. The macroelements (S, P, K, Mg, and Fe) and Ca were, respectively, correlated closely with the alpha diversity of prokaryotes and fungi, while the macronutrients (e.g., Na, S, P, and Ca) were correlated with the prokaryotic and fungal beta-diversity ( P ≤ 0.05). And, the nine microelements (e.g., Al, Ba, Co, Hg, and Mn) and micronutrients (Ba, Cd, and Sr) individually shaped the alpha diversity of prokaryotes and fungi, while the six microelements (e.g., As, Ba, Cr, and Ge) and only the trace elements (Cr and Cu), respectively, influenced the beta diversity of prokaryotes and fungi ( P analysis (VPA) showed that environmental variables jointly explained 55.49% and 32.27% of the total variation for the prokaryotic and fungal communities, respectively. Together, our findings demonstrate that the diversity and community composition of the prokaryotes and fungi were driven by different macro and microelements in saline habitats, and that geochemical elements could more widely regulate the diversity and community composition of prokaryotes than these of fungi.

Macro and Microelements Drive Diversity and Composition of Prokaryotic and Fungal Communities in Hypersaline Sediments and Saline–Alkaline Soils

Science.gov (United States)

Liu, Kaihui; Ding, Xiaowei; Tang, Xiaofei; Wang, Jianjun; Li, Wenjun; Yan, Qingyun; Liu, Zhenghua

2018-01-01

Understanding the effects of environmental factors on microbial communities is critical for microbial ecology, but it remains challenging. In this study, we examined the diversity (alpha diversity) and community compositions (beta diversity) of prokaryotes and fungi in hypersaline sediments and salinized soils from northern China. Environmental variables were highly correlated, but they differed significantly between the sediments and saline soils. The compositions of prokaryotic and fungal communities in the hypersaline sediments were different from those in adjacent saline–alkaline soils, indicating a habitat-specific microbial distribution pattern. The macroelements (S, P, K, Mg, and Fe) and Ca were, respectively, correlated closely with the alpha diversity of prokaryotes and fungi, while the macronutrients (e.g., Na, S, P, and Ca) were correlated with the prokaryotic and fungal beta-diversity (P ≤ 0.05). And, the nine microelements (e.g., Al, Ba, Co, Hg, and Mn) and micronutrients (Ba, Cd, and Sr) individually shaped the alpha diversity of prokaryotes and fungi, while the six microelements (e.g., As, Ba, Cr, and Ge) and only the trace elements (Cr and Cu), respectively, influenced the beta diversity of prokaryotes and fungi (P analysis (VPA) showed that environmental variables jointly explained 55.49% and 32.27% of the total variation for the prokaryotic and fungal communities, respectively. Together, our findings demonstrate that the diversity and community composition of the prokaryotes and fungi were driven by different macro and microelements in saline habitats, and that geochemical elements could more widely regulate the diversity and community composition of prokaryotes than these of fungi. PMID:29535703

Transcriptional Silencing of Retroviral Vectors

DEFF Research Database (Denmark)

Lund, Anders Henrik; Duch, M.; Pedersen, F.S.

1996-01-01

. Extinction of long-term vector expression has been observed after implantation of transduced hematopoietic cells as well as fibroblasts, myoblasts and hepatocytes. Here we review the influence of vector structure, integration site and cell type on transcriptional silencing. While down-regulation of proviral...... transcription is known from a number of cellular and animal models, major insight has been gained from studies in the germ line and embryonal cells of the mouse. Key elements for the transfer and expression of retroviral vectors, such as the viral transcriptional enhancer and the binding site for the t......RNA primer for reverse transcription may have a major influence on transcriptional silencing. Alterations of these elements of the vector backbone as well as the use of internal promoter elements from housekeeping genes may contribute to reduce transcriptional silencing. The use of cell culture and animal...

Cloning and Expression of Leptospira LipL32 Antigen as a Candidate for Rapid Diagnosis

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Nooshin Sohrabi

2013-09-01

Full Text Available Background and Objective: Leptospirosis as an important emerging infectious zoonotic disease caused by spirochetes of the genus Leptospira. Given the low sensitivity and long duration of its culture, the diagnosis of leptospirosis is mainly based on serological methods. The microscopic agglutination test (MAT is considered as the reference method. Because of the complexity of the MAT, there is an urgent need for the development of new reliable and rapid screening tests for leptospirosis. Major leptospiral outer membrane proteins (OMPs, present only in pathologic strains, could be regarded as a good candidate for diagnostic studies. Here we report the cloning and expression of LipL32, as a prominent immunogenic protein, in a prokaryotic system. Materials and Methods: After the amplification of LipL32 gene, it was cloned into the pQE30 vector. The insertion of LipL32 gene into the vector was screened and confirmed with restriction analysis and sequencing. The recombinant plasmid was transformed into E. coli M15 strain, and the expressed protein was identified by SDS-PAGE and western blotting. This recombinant protein with 6× His-tagged sequence was purified using Ni-NTA affinity column chromatography. Results: The results revealed that the selected gene was successfully cloned in pQE30 vector and recombinant protein (rLipL32 of approximately ~32 kDa was produced, purified and confirmed by western blotting. Conclusion: This recombinant protein could be potentially used for the development of serodiagnosis tests for the diagnosis of leptospirosis in humans and animals.

Molecular characterisation and expression analysis of acc oxidase gene from guzmania ruiz and pav

International Nuclear Information System (INIS)

Jianxin, L.; Huaqiao, D.; Weiyong, W.; Danqing, T.

2017-01-01

ACC oxidase is the last key enzyme of ethylene synthesis pathway, while ethylene is a key factor affecting flowering in ornamental bromeliad. To understand ACC oxidase gene's characteristics and its effect on ornamental bromeliad flowering, we cloned 1504bp full-length cDNA sequence (GenBank: JX972145) and 2546bp corresponding genomic sequence (GenBank: JX972146)of GoACO1 (ACC oxidase gene) from Guzmania variety: Ostara. Prokaryotic expression study showed that expression of GoACO1 can produced a 41 KD protein precipitation in Escherichia coli DE3(BL-21); Real-time quantitative analysis showed that GoACO1 can express in all tested tissues including floral organ, bract, leaf and scape, and expression quantity in bract was the highest. Through constructing plant overexpression vector, transforming into Arabidopsis thaliana, and investigating blossom character of T2 generation seeds, we found that first flowering time of the goal Arabidopsis thaliana was 1.5 days earlier, and their peak flowering time(the number of flowering more than 50%) was 1.8 days earlier, compared with wild type one. Taken together, our results suggested that GoACO1can express in all kinds of tissues and seems to promote Arabidopsis thaliana flowering earlier. (author)

Versatile Cas9-driven subpopulation selection toolbox for Lactococcus lactis

NARCIS (Netherlands)

Els, van der Simon; James, Jennelle K.; Kleerebezem, Michiel; Bron, Peter A.

2018-01-01

CRISPR-Cas9 technology has been exploited for the removal or replacement of genetic elements in a wide range of prokaryotes and eukaryotes. Here, we describe the extension of the Cas9 application toolbox to the industrially important dairy species Lactococcus lactis. The Cas9 expression vector

Symbolic computer vector analysis

Science.gov (United States)

Stoutemyer, D. R.

1977-01-01

A MACSYMA program is described which performs symbolic vector algebra and vector calculus. The program can combine and simplify symbolic expressions including dot products and cross products, together with the gradient, divergence, curl, and Laplacian operators. The distribution of these operators over sums or products is under user control, as are various other expansions, including expansion into components in any specific orthogonal coordinate system. There is also a capability for deriving the scalar or vector potential of a vector field. Examples include derivation of the partial differential equations describing fluid flow and magnetohydrodynamics, for 12 different classic orthogonal curvilinear coordinate systems.

Links between viruses and prokaryotes throughout the water column along a North Atlantic latitudinal transect

NARCIS (Netherlands)

De Corte, Daniele; Sintes, Eva; Yokokawa, Taichi; Reinthaler, Thomas; Herndl, Gerhard J.

Viruses are an abundant, diverse and dynamic component of marine ecosystems and have a key role in the biogeochemical processes of the ocean by controlling prokaryotic and phytoplankton abundance and diversity. However, most of the studies on virus-prokaryote interactions in marine environments have

Rumen prokaryotic communities of ruminants under different feeding paradigms on the Qinghai-Tibetan Plateau.

Science.gov (United States)

Xue, Dan; Chen, Huai; Zhao, Xinquan; Xu, Shixiao; Hu, Linyong; Xu, Tianwei; Jiang, Lin; Zhan, Wei

2017-06-01

Yak and Tibetan sheep are the major indigenous ruminants on the Qinghai-Tibetan Plateau in China. The aim of this work was to study the differences in ruminal fermentation parameters and rumen prokaryotic community composition between hosts and feeding paradigms. The 16S rRNA genes targeting bacteria and archaea were sequenced using the MiSeq platform. The results showed that the prokaryotic community structure between yak and Tibetan sheep was significantly different (PTibetan sheep of the two groups (P=0.026). The core prokaryotic populations that existed in the rumen mostly dominated the structure. There was an obvious correlation of the prokaryotic community composition at the phylum and genus levels with the host or the feeding pattern. In addition, Tibetan sheep showed significantly higher yields of volatile fatty acids (VFAs) than yak, as did the NG group compared with the TMR group. In conclusion, both the host and feeding pattern may influence rumen microbial ecology system, with host effects being more important than those of the feeding pattern. Copyright © 2017 Elsevier GmbH. All rights reserved.

Lentiviral Vector Gene Transfer to Porcine Airways

Directory of Open Access Journals (Sweden)

Patrick L Sinn

2012-01-01

Full Text Available In this study, we investigated lentiviral vector development and transduction efficiencies in well-differentiated primary cultures of pig airway epithelia (PAE and wild-type pigs in vivo. We noted gene transfer efficiencies similar to that observed for human airway epithelia (HAE. Interestingly, feline immunodeficiency virus (FIV-based vectors transduced immortalized pig cells as well as pig primary cells more efficiently than HIV-1–based vectors. PAE express TRIM5α, a well-characterized species-specific lentiviral restriction factor. We contrasted the restrictive properties of porcine TRIM5α against FIV- and HIV-based vectors using gain and loss of function approaches. We observed no effect on HIV-1 or FIV conferred transgene expression in response to porcine TRIM5α overexpression or knockdown. To evaluate the ability of GP64-FIV to transduce porcine airways in vivo, we delivered vector expressing mCherry to the tracheal lobe of the lung and the ethmoid sinus of 4-week-old pigs. One week later, epithelial cells expressing mCherry were readily detected. Our findings indicate that pseudotyped FIV vectors confer similar tropisms in porcine epithelia as observed in human HAE and provide further support for the selection of GP64 as an appropriate envelope pseudotype for future preclinical gene therapy studies in the porcine model of cystic fibrosis (CF.

Small CRISPR RNAs guide antiviral defense in prokaryotes

NARCIS (Netherlands)

Brouns, S.J.J.; Jore, M.M.; Lundgren, M.; Westra, E.R.; Slijkhuis, R.J.; Snijders, A.P.; Dickman, M.J.; Makarova, K.S.; Koonin, E.V.; Oost, van der J.

2008-01-01

Prokaryotes acquire virus resistance by integrating short fragments of viral nucleic acid into clusters of regularly interspaced short palindromic repeats (CRISPRs). Here we show how virus-derived sequences contained in CRISPRs are used by CRISPR-associated (Cas) proteins from the host to mediate an

Surveying the expanding prokaryotic Rubisco multiverse.

Science.gov (United States)

Liu, Di; Ramya, Ramaswamy Chettiyan Seetharaman; Mueller-Cajar, Oliver

2017-09-01

The universal, but catalytically modest, CO2-fixing enzyme Rubisco is currently experiencing intense interest by researchers aiming to enhance crop photosynthesis. These efforts are mostly focused on the highly conserved hexadecameric enzyme found in land plants. In comparison, prokaryotic organisms harbor a far greater diversity in Rubisco forms. Recent work towards improving our appreciation of microbial Rubisco properties and harnessing their potential is surveyed. New structural models are providing informative glimpses into catalytic subtleties and diverse oligomeric states. Ongoing characterization is informing us about the conservation of constraints, such as sugar phosphate inhibition and the associated dependence on Rubisco activase helper proteins. Prokaryotic Rubiscos operate under a far wider range of metabolic contexts than the photosynthetic function of higher plant enzymes. Relaxed selection pressures may have resulted in the exploration of a larger volume of sequence space than permitted in organisms performing oxygenic photosynthesis. To tap into the potential of microbial Rubiscos, in vivo selection systems are being used to discover functional metagenomic Rubiscos. Various directed evolution systems to optimize their function have been developed. It is anticipated that this approach will provide access to biotechnologically valuable enzymes that cannot be encountered in the higher plant Rubisco space. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Trends and barriers to lateral gene transfer in prokaryotes.

Science.gov (United States)

Popa, Ovidiu; Dagan, Tal

2011-10-01

Gene acquisition by lateral gene transfer (LGT) is an important mechanism for natural variation among prokaryotes. Laboratory experiments show that protein-coding genes can be laterally transferred extremely fast among microbial cells, inherited to most of their descendants, and adapt to a new regulatory regime within a short time. Recent advance in the phylogenetic analysis of microbial genomes using networks approach reveals a substantial impact of LGT during microbial genome evolution. Phylogenomic networks of LGT among prokaryotes reconstructed from completely sequenced genomes uncover barriers to LGT in multiple levels. Here we discuss the kinds of barriers to gene acquisition in nature including physical barriers for gene transfer between cells, genomic barriers for the integration of acquired DNA, and functional barriers for the acquisition of new genes. Copyright © 2011 Elsevier Ltd. All rights reserved.

aeGEPUCI: a database of gene expression in the dengue vector mosquito, Aedes aegypti

Directory of Open Access Journals (Sweden)

James Anthony A

2010-10-01

Full Text Available Abstract Background Aedes aegypti is the principal vector of dengue and yellow fever viruses. The availability of the sequenced and annotated genome enables genome-wide analyses of gene expression in this mosquito. The large amount of data resulting from these analyses requires efficient cataloguing before it becomes useful as the basis for new insights into gene expression patterns and studies of the underlying molecular mechanisms for generating these patterns. Findings We provide a publicly-accessible database and data-mining tool, aeGEPUCI, that integrates 1 microarray analyses of sex- and stage-specific gene expression in Ae. aegypti, 2 functional gene annotation, 3 genomic sequence data, and 4 computational sequence analysis tools. The database can be used to identify genes expressed in particular stages and patterns of interest, and to analyze putative cis-regulatory elements (CREs that may play a role in coordinating these patterns. The database is accessible from the address http://www.aegep.bio.uci.edu. Conclusions The combination of gene expression, function and sequence data coupled with integrated sequence analysis tools allows for identification of expression patterns and streamlines the development of CRE predictions and experiments to assess how patterns of expression are coordinated at the molecular level.

VEST: Abstract vector calculus simplification in Mathematica

Science.gov (United States)

Squire, J.; Burby, J.; Qin, H.

2014-01-01

We present a new package, VEST (Vector Einstein Summation Tools), that performs abstract vector calculus computations in Mathematica. Through the use of index notation, VEST is able to reduce three-dimensional scalar and vector expressions of a very general type to a well defined standard form. In addition, utilizing properties of the Levi-Civita symbol, the program can derive types of multi-term vector identities that are not recognized by reduction, subsequently applying these to simplify large expressions. In a companion paper Burby et al. (2013) [12], we employ VEST in the automation of the calculation of high-order Lagrangians for the single particle guiding center system in plasma physics, a computation which illustrates its ability to handle very large expressions. VEST has been designed to be simple and intuitive to use, both for basic checking of work and more involved computations.

Particle-association lifestyle is a phylogenetically conserved trait in bathypelagic prokaryotes

KAUST Repository

Salazar, Guillem; Cornejo-Castillo, Francisco M.; Borrull, Encarna; Dí ez-Vives, Cristina; Lara, Elena; Vaqué , Dolors; Arrieta, J M; Duarte, Carlos M.; Gasol, Josep M.; Acinas, Silvia G.

2015-01-01

The free-living (FL) and particle-attached (PA) marine microbial communities have repeatedly been proved to differ in their diversity and composition in the photic ocean and also recently in the bathypelagic ocean at a global scale. However, although high taxonomic ranks exhibit preferences for a PA or FL mode of life, it remains poorly understood whether two clear lifestyles do exist and how these are distributed across the prokaryotic phylogeny. We studied the FL (<0.8 μm) and PA (0.8 – 20 μm) prokaryotes at 30 stations distributed worldwide within the bathypelagic oceanic realm (2,150 – 4,000 m depth) using high throughput sequencing of the small subunit ribosomal RNA gene (16S rRNA). A high proportion of the bathypelagic prokaryotes were mostly found either attached to particles or freely in the surrounding water but rarely in both types of environments. In particular, this trait was deeply conserved through their phylogeny suggesting that the deep-ocean particles and the surrounding water constitute two highly distinct niches and that transitions from one to the other have been rare at an evolutionary time-scale. As a consequence, PA and FL communities had clear alpha- and beta-diversity differences that exceeded the global-scale geographical variation. Our study organizes the bathypelagic prokaryotic diversity into a reasonable number of ecologically coherent taxa regarding their association to particles, a first step for understanding which are the microbes responsible for the processing of the dissolved and particulate pools of organic matter that have a very different biogeochemical role in the deep ocean.

Particle-association lifestyle is a phylogenetically conserved trait in bathypelagic prokaryotes

KAUST Repository

Salazar, Guillem

2015-10-13

The free-living (FL) and particle-attached (PA) marine microbial communities have repeatedly been proved to differ in their diversity and composition in the photic ocean and also recently in the bathypelagic ocean at a global scale. However, although high taxonomic ranks exhibit preferences for a PA or FL mode of life, it remains poorly understood whether two clear lifestyles do exist and how these are distributed across the prokaryotic phylogeny. We studied the FL (<0.8 μm) and PA (0.8 – 20 μm) prokaryotes at 30 stations distributed worldwide within the bathypelagic oceanic realm (2,150 – 4,000 m depth) using high throughput sequencing of the small subunit ribosomal RNA gene (16S rRNA). A high proportion of the bathypelagic prokaryotes were mostly found either attached to particles or freely in the surrounding water but rarely in both types of environments. In particular, this trait was deeply conserved through their phylogeny suggesting that the deep-ocean particles and the surrounding water constitute two highly distinct niches and that transitions from one to the other have been rare at an evolutionary time-scale. As a consequence, PA and FL communities had clear alpha- and beta-diversity differences that exceeded the global-scale geographical variation. Our study organizes the bathypelagic prokaryotic diversity into a reasonable number of ecologically coherent taxa regarding their association to particles, a first step for understanding which are the microbes responsible for the processing of the dissolved and particulate pools of organic matter that have a very different biogeochemical role in the deep ocean.

Connectivity between surface and deep waters determines prokaryotic diversity in the North Atlantic Deep Water.

Science.gov (United States)

Frank, Alexander H; Garcia, Juan A L; Herndl, Gerhard J; Reinthaler, Thomas

2016-06-01

To decipher the influence of depth stratification and surface provincialism on the dark ocean prokaryotic community composition, we sampled the major deep-water masses in the eastern North Atlantic covering three biogeographic provinces. Their diversity was evaluated using ordination and canonical analysis of 454 pyrotag sequences. Variance partitioning suggested that 16% of the variation in the bacterial community composition was based on depth stratification while 9% of the variation was due to geographic location. General linear mixed effect models showed that the community of the subsurface waters was connected to the dark ocean prokaryotic communities in different biogeographic provinces. Cluster analysis indicated that some prokaryotic taxa are specific to distinct regions in bathypelagic water masses. Taken together, our data suggest that the dark ocean prokaryotic community composition of the eastern North Atlantic is primed by the formation and the horizontal transport of water masses. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

A cryptic promoter in potato virus X vector interrupted plasmid construction

Directory of Open Access Journals (Sweden)

Schultz Ronald D

2007-03-01

Full Text Available Abstract Background Potato virus X has been developed into an expression vector for plants. It is widely used to express foreign genes. In molecular manipulation, the foreign genes need to be sub-cloned into the vector. The constructed plasmid needs to be amplified. Usually, during amplification stage, the foreign genes are not expressed. However, if the foreign gene is expressed, the construction work could be interrupted. Two different viral genes were sub-cloned into the vector, but only one foreign gene was successfully sub-cloned. The other foreign gene, canine parvovirus type 2 (CPV-2 VP1 could not be sub-cloned into the vector and amplified without mutation (frame shift mutation. Results A cryptic promoter in the PVX vector was discovered with RT-PCR. The promoter activity was studied with Northern blots and Real-time RT-PCR. Conclusion It is important to recognize the homologous promoter sequences in the vector when a virus is developed as an expression vector. During the plasmid amplification stage, an unexpected expression of the CPV-2 VP1 gene (not in the target plants, but in E. coli can interrupt the downstream work.

Molecular Characterization of Heterologous HIV-1gp120 Gene Expression Disruption in Mycobacterium bovis BCG Host Strain: A Critical Issue for Engineering Mycobacterial Based-Vaccine Vectors

Science.gov (United States)

Joseph, Joan; Fernández-Lloris, Raquel; Pezzat, Elías; Saubi, Narcís; Cardona, Pere-Joan; Mothe, Beatriz; Gatell, Josep Maria

2010-01-01

Mycobacterium bovis Bacillus Calmette-Guérin (BCG) as a live vector of recombinant bacterial vaccine is a promising system to be used. In this study, we evaluate the disrupted expression of heterologous HIV-1gp120 gene in BCG Pasteur host strain using replicative vectors pMV261 and pJH222. pJH222 carries a lysine complementing gene in BCG lysine auxotrophs. The HIV-1 gp120 gene expression was regulated by BCG hsp60 promoter (in plasmid pMV261) and Mycobacteria spp. α-antigen promoter (in plasmid pJH222). Among 14 rBCG:HIV-1gp120 (pMV261) colonies screened, 12 showed a partial deletion and two showed a complete deletion. However, deletion was not observed in all 10 rBCG:HIV-1gp120 (pJH222) colonies screened. In this study, we demonstrated that E. coli/Mycobacterial expression vectors bearing a weak promoter and lysine complementing gene in a recombinant lysine auxotroph of BCG could prevent genetic rearrangements and disruption of HIV 1gp120 gene expression, a key issue for engineering Mycobacterial based vaccine vectors. PMID:20617151

Molecular Characterization of Heterologous HIV-1gp120 Gene Expression Disruption in Mycobacterium bovis BCG Host Strain: A Critical Issue for Engineering Mycobacterial Based-Vaccine Vectors

Directory of Open Access Journals (Sweden)

Joan Joseph

2010-01-01

Full Text Available Mycobacterium bovis Bacillus Calmette-Guérin (BCG as a live vector of recombinant bacterial vaccine is a promising system to be used. In this study, we evaluate the disrupted expression of heterologous HIV-1gp120 gene in BCG Pasteur host strain using replicative vectors pMV261 and pJH222. pJH222 carries a lysine complementing gene in BCG lysine auxotrophs. The HIV-1 gp120 gene expression was regulated by BCG hsp60 promoter (in plasmid pMV261 and Mycobacteria spp. α-antigen promoter (in plasmid pJH222. Among 14 rBCG:HIV-1gp120 (pMV261 colonies screened, 12 showed a partial deletion and two showed a complete deletion. However, deletion was not observed in all 10 rBCG:HIV-1gp120 (pJH222 colonies screened. In this study, we demonstrated that E. coli/Mycobacterial expression vectors bearing a weak promoter and lysine complementing gene in a recombinant lysine auxotroph of BCG could prevent genetic rearrangements and disruption of HIV 1gp120 gene expression, a key issue for engineering Mycobacterial based vaccine vectors.

A new strategy for full-length Ebola virus glycoprotein expression in E.coli.

Science.gov (United States)

Zai, Junjie; Yi, Yinhua; Xia, Han; Zhang, Bo; Yuan, Zhiming

2016-12-01

Ebola virus (EBOV) causes severe hemorrhagic fever in humans and non-human primates with high rates of fatality. Glycoprotein (GP) is the only envelope protein of EBOV, which may play a critical role in virus attachment and entry as well as stimulating host protective immune responses. However, the lack of expression of full-length GP in Escherichia coli hinders the further study of its function in viral pathogenesis. In this study, the vp40 gene was fused to the full-length gp gene and cloned into a prokaryotic expression vector. We showed that the VP40-GP and GP-VP40 fusion proteins could be expressed in E.coli at 16 °C. In addition, it was shown that the position of vp40 in the fusion proteins affected the yields of the fusion proteins, with a higher level of production of the fusion protein when vp40 was upstream of gp compared to when it was downstream. The results provide a strategy for the expression of a large quantity of EBOV full-length GP, which is of importance for further analyzing the relationship between the structure and function of GP and developing an antibody for the treatment of EBOV infection.

Prosecutor: parameter-free inference of gene function for prokaryotes using DNA microarray data, genomic context and multiple gene annotation sources

Directory of Open Access Journals (Sweden)

van Hijum Sacha AFT

2008-10-01

Full Text Available Abstract Background Despite a plethora of functional genomic efforts, the function of many genes in sequenced genomes remains unknown. The increasing amount of microarray data for many species allows employing the guilt-by-association principle to predict function on a large scale: genes exhibiting similar expression patterns are more likely to participate in shared biological processes. Results We developed Prosecutor, an application that enables researchers to rapidly infer gene function based on available gene expression data and functional annotations. Our parameter-free functional prediction method uses a sensitive algorithm to achieve a high association rate of linking genes with unknown function to annotated genes. Furthermore, Prosecutor utilizes additional biological information such as genomic context and known regulatory mechanisms that are specific for prokaryotes. We analyzed publicly available transcriptome data sets and used literature sources to validate putative functions suggested by Prosecutor. We supply the complete results of our analysis for 11 prokaryotic organisms on a dedicated website. Conclusion The Prosecutor software and supplementary datasets available at http://www.prosecutor.nl allow researchers working on any of the analyzed organisms to quickly identify the putative functions of their genes of interest. A de novo analysis allows new organisms to be studied.

RNA-seq analyses of blood-induced changes in gene expression in the mosquito vector species, Aedes aegypti

Directory of Open Access Journals (Sweden)

Olson Ken E

2011-01-01

Full Text Available Abstract Background Hematophagy is a common trait of insect vectors of disease. Extensive genome-wide transcriptional changes occur in mosquitoes after blood meals, and these are related to digestive and reproductive processes, among others. Studies of these changes are expected to reveal molecular targets for novel vector control and pathogen transmission-blocking strategies. The mosquito Aedes aegypti (Diptera, Culicidae, a vector of Dengue viruses, Yellow Fever Virus (YFV and Chikungunya virus (CV, is the subject of this study to look at genome-wide changes in gene expression following a blood meal. Results Transcriptional changes that follow a blood meal in Ae. aegypti females were explored using RNA-seq technology. Over 30% of more than 18,000 investigated transcripts accumulate differentially in mosquitoes at five hours after a blood meal when compared to those fed only on sugar. Forty transcripts accumulate only in blood-fed mosquitoes. The list of regulated transcripts correlates with an enhancement of digestive activity and a suppression of environmental stimuli perception and innate immunity. The alignment of more than 65 million high-quality short reads to the Ae. aegypti reference genome permitted the refinement of the current annotation of transcript boundaries, as well as the discovery of novel transcripts, exons and splicing variants. Cis-regulatory elements (CRE and cis-regulatory modules (CRM enriched significantly at the 5'end flanking sequences of blood meal-regulated genes were identified. Conclusions This study provides the first global view of the changes in transcript accumulation elicited by a blood meal in Ae. aegypti females. This information permitted the identification of classes of potentially co-regulated genes and a description of biochemical and physiological events that occur immediately after blood feeding. The data presented here serve as a basis for novel vector control and pathogen transmission

Adeno-associated viral vector serotypes 1 and 5 targeted to the neonatal rat and pig striatum induce widespread transgene expression in the forebrain

DEFF Research Database (Denmark)

Kornum, Birgitte R; Stott, Simon R W; Mattsson, Bengt

2010-01-01

. Our results show that striatal delivery of rAAV5 vectors in the neonatal brain represents a useful tool to express genes of interest both in the basal ganglia and the neocortex. Furthermore, we apply, for the first time, viral vector-mediated gene transfer to the pig brain providing the opportunity...

Evaluation of a vectored equine herpesvirus type 1 (EHV-1) vaccine expressing H3 haemagglutinin in the protection of dogs against canine influenza

OpenAIRE

Rosas, Cristina; Van de Walle, Gerlinde R.; Metzger, Stephan M.; Hoelzer, Karin; Dubovi, Edward J.; Kim, Sung G.; Parrish, Colin R.; Osterrieder, Nikolaus

2008-01-01

In 2004, canine influenza virus (CIV) was identified as a respiratory pathogen of dogs for the first time and is closely related to H3N8 equine influenza virus (EIV). We generated a recombinant vectored vaccine that expresses H3 of a recent isolate of EIV using equine herpesvirus type 1 (EHV-1) as the delivery vehicle. This EHV-1 vectored vaccine exhibited robust and stable EIV H3 expression and induced a strong influenza virus-specific response in both mice and dogs upon intranasal or subcut...

Rule Mining Techniques to Predict Prokaryotic Metabolic Pathways

KAUST Repository

Saidi, Rabie

2017-08-28

It is becoming more evident that computational methods are needed for the identification and the mapping of pathways in new genomes. We introduce an automatic annotation system (ARBA4Path Association Rule-Based Annotator for Pathways) that utilizes rule mining techniques to predict metabolic pathways across wide range of prokaryotes. It was demonstrated that specific combinations of protein domains (recorded in our rules) strongly determine pathways in which proteins are involved and thus provide information that let us very accurately assign pathway membership (with precision of 0.999 and recall of 0.966) to proteins of a given prokaryotic taxon. Our system can be used to enhance the quality of automatically generated annotations as well as annotating proteins with unknown function. The prediction models are represented in the form of human-readable rules, and they can be used effectively to add absent pathway information to many proteins in UniProtKB/TrEMBL database.

Rule Mining Techniques to Predict Prokaryotic Metabolic Pathways

KAUST Repository

Saidi, Rabie; Boudellioua, Imene; Martin, Maria J.; Solovyev, Victor

2017-01-01

It is becoming more evident that computational methods are needed for the identification and the mapping of pathways in new genomes. We introduce an automatic annotation system (ARBA4Path Association Rule-Based Annotator for Pathways) that utilizes rule mining techniques to predict metabolic pathways across wide range of prokaryotes. It was demonstrated that specific combinations of protein domains (recorded in our rules) strongly determine pathways in which proteins are involved and thus provide information that let us very accurately assign pathway membership (with precision of 0.999 and recall of 0.966) to proteins of a given prokaryotic taxon. Our system can be used to enhance the quality of automatically generated annotations as well as annotating proteins with unknown function. The prediction models are represented in the form of human-readable rules, and they can be used effectively to add absent pathway information to many proteins in UniProtKB/TrEMBL database.

Expression of the human blood coagulation protein factor XIIIa in Saccharomyces cerevisiae: dependence of the expression levels from host-vector systems and medium conditions.

Science.gov (United States)

Bröker, M; Bäuml, O; Göttig, A; Ochs, J; Bodenbenner, M; Amann, E

1991-03-01

The human blood coagulation protein Factor XIIIa (FXIIIa) was expressed in Saccharomyces cerevisiae employing Escherichia coli-yeast shuttle vectors based on a 2-mu plasmid. Several factors affecting high production yield of recombinant FXIIIa were analysed. The use of the regulatable GAL-CYC1 hybrid promoter resulted in higher FXIIIa expression when compared with the constitutive ADCI promoter. Screening for suitable yeast strains for expression of FXIIIa under the transcriptional control of the GAL-CYC1 hybrid promoter revealed a broad spectrum of productivity. No obvious correlation between the expression rate and the genetic markers of the strains could be identified. The medium composition markedly influenced the FXIIIa expression rates. The expression of FXIIIa was strictly regulated by the carbon source. Glucose as the only sugar and energy source repressed the synthesis of FXIIIa, whereas addition of galactose induced FXIIIa expression. Special feeding schemes resulted in a productivity of up to 100 mg FXIIIa/l in shake flasks.

Genomics of bacteria and archaea: the emerging dynamic view of the prokaryotic world

Science.gov (United States)

Koonin, Eugene V.; Wolf, Yuri I.

2008-01-01

The first bacterial genome was sequenced in 1995, and the first archaeal genome in 1996. Soon after these breakthroughs, an exponential rate of genome sequencing was established, with a doubling time of approximately 20 months for bacteria and approximately 34 months for archaea. Comparative analysis of the hundreds of sequenced bacterial and dozens of archaeal genomes leads to several generalizations on the principles of genome organization and evolution. A crucial finding that enables functional characterization of the sequenced genomes and evolutionary reconstruction is that the majority of archaeal and bacterial genes have conserved orthologs in other, often, distant organisms. However, comparative genomics also shows that horizontal gene transfer (HGT) is a dominant force of prokaryotic evolution, along with the loss of genetic material resulting in genome contraction. A crucial component of the prokaryotic world is the mobilome, the enormous collection of viruses, plasmids and other selfish elements, which are in constant exchange with more stable chromosomes and serve as HGT vehicles. Thus, the prokaryotic genome space is a tightly connected, although compartmentalized, network, a novel notion that undermines the ‘Tree of Life’ model of evolution and requires a new conceptual framework and tools for the study of prokaryotic evolution. PMID:18948295

Adenoviral vector-mediated gene expression in the nervous system of immunocompetent Wistar and T cell-deficient nude rats : preferential survival of transduced astroglial cells in nude rats

NARCIS (Netherlands)

Hermens, W.T.J.M.C.; Verhaagen, J

1997-01-01

In the present paper, we examined the effect of the adenoviral vector dosage, the role of T cells, and the influence of the presence of replication-competent adenovirus (RCA) in adenoviral vector stocks, on the efficacy of adenoviral vector-directed transgene expression in the facial nucleus of

A new adenovirus based vaccine vector expressing an Eimeria tenella derived TLR agonist improves cellular immune responses to an antigenic target.

Directory of Open Access Journals (Sweden)

Daniel M Appledorn

2010-03-01

Full Text Available Adenoviral based vectors remain promising vaccine platforms for use against numerous pathogens, including HIV. Recent vaccine trials utilizing Adenovirus based vaccines expressing HIV antigens confirmed induction of cellular immune responses, but these responses failed to prevent HIV infections in vaccinees. This illustrates the need to develop vaccine formulations capable of generating more potent T-cell responses to HIV antigens, such as HIV-Gag, since robust immune responses to this antigen correlate with improved outcomes in long-term non-progressor HIV infected individuals.In this study we designed a novel vaccine strategy utilizing an Ad-based vector expressing a potent TLR agonist derived from Eimeria tenella as an adjuvant to improve immune responses from a [E1-]Ad-based HIV-Gag vaccine. Our results confirm that expression of rEA elicits significantly increased TLR mediated innate immune responses as measured by the influx of plasma cytokines and chemokines, and activation of innate immune responding cells. Furthermore, our data show that the quantity and quality of HIV-Gag specific CD8(+ and CD8(- T-cell responses were significantly improved when coupled with rEA expression. These responses also correlated with a significantly increased number of HIV-Gag derived epitopes being recognized by host T cells. Finally, functional assays confirmed that rEA expression significantly improved antigen specific CTL responses, in vivo. Moreover, we show that these improved responses were dependent upon improved TLR pathway interactions.The data presented in this study illustrate the potential utility of Ad-based vectors expressing TLR agonists to improve clinical outcomes dependent upon induction of robust, antigen specific immune responses.

[Expression in E.coli and bioactivity assay of Micrococcus luteus resuscitation promoting factor domain and its mutants].

Science.gov (United States)

Yue, Chen-Li; Shi, Jie-Ran; Shi, Chang-Hong; Zhang, Hai; Zhao, Lei; Zhang, Ting-Fen; Zhao, Yong; Xi, Li

2008-10-01

To express Micrococcus luteus resuscitation promoting factor (Rpf) domain and its mutants in prokaryotic cells, and to investigate their bioactivity. The gene of Rpf domain and its mutants (E54K, E54A) were amplified by polymerase chain reaction (PCR) from the genome of Micrococcus luteus and cloned into pMD18-T vector. After sequenced, the Rpf domain and its mutant gene were subcloned into expression vector PGEX-4T-1, and transfected into E. coli DH5alpha. The expressed product was purified by affinity chromatography using GST Fusion Protein Purification bead. The aim proteins were identified by SDS-PAGE analysis and by Western blot with monoclonal antibodies against Rpf domain (mAb). The bioactivity of the proteins was analyzed by stimulating the resuscitation of Mycobacterium smegmatis. The sequences of the PCR products were identical to those of the Rpf domain and its mutant gene in GenBank. The relative molecular mass identified by SDS-PAGE analysis was consistent with that had been reported, which was also confirmed by Western blot analysis that there were specific bindings at 32 000 with Rpf domain mAb. The purified GST-Rpf domain could stimulate resuscitation of Mycobacterium smegmatis. Replacements E54A and especially E54K resulted in inhibition of Rpf resuscitation activity. Rpf domain and two kinds of its mutant protein were obtained, and its effects on the resuscitation of dormant Mycobacterium smegmatis were clarified.

Tissue-specific expression of silkmoth chorion genes in vivo using Bombyx mori nuclear polyhedrosis virus as a transducing vector.

Science.gov (United States)

Iatrou, K; Meidinger, R G

1990-01-01

A pair of silkmoth chorion chromosomal genes, HcA.12-HcB.12, was inserted into a baculovirus transfer vector, pBmp2, derived from the nuclear polyhedrosis virus of Bombyx mori. This vector, which permits the insertion of foreign genetic material in the vicinity of a mutationally inactivated polyhedrin gene, was used to acquire the corresponding recombinant virus. Injection of mutant silkmoth pupae that lack all Hc chorion genes with the recombinant virus resulted in the infection of all internal organs including follicular tissue. Analysis of RNA from infected tissues has demonstrated that the two chorion genes present in the viral genome are correctly transcribed under the control of their own promoter in follicular cells, the tissue in which chorion genes are normally expressed. The chorion primary transcripts are also correctly processed in the infected follicular cells and yield mature mRNAs indistinguishable from authentic chorion mRNAs present in wild-type follicles. These results demonstrate that recombinant nuclear polyhedrosis viruses can be used as transducing vectors for introducing genetic material of host origin into the cells of the organism and that the transduced genes are transiently expressed in a tissue-specific manner under the control of their resident regulatory sequences. Thus we show the in vivo expression of cloned genes under cellular promoter control in an insect other than Drosophila melanogaster. The approach should be applicable to all insect systems that are subject to nuclear polyhedrosis virus infection. Images PMID:2187186

Short Term INT-Formazan Production as a Proxy for Marine Prokaryote Respiration

Science.gov (United States)

Cajal-Medrano, R.; Villegas-Mendoza, J.; Maske, H.

2016-02-01

Prokaryotes are poisoned by the tetrazolium electron transport probe INT on time scales of less than one hour, invalidating the interpretation of the rate of in vivo INT reduction to formazan as a proxy for oxygen consumption rates (Villegas-Mendoza et al. 2015). We measured oxygen consumption rate (R; µM O2 hour-1) and electron transport activity with in vivo INT formazan production (IFP, mM formazan) at 0.5 mM INT during 1 hour exposure time of natural communities and cultures of the marine bacteria Vibrio harveyi growing in batch and continuous cultures. A strong exponential relationship R = 0.20 IFP2.15 (pgrowth rates under aerobic condition. We find that IFP and oxygen consumption increase with bacterial specific growth rates and temperature as expected from basic principles of physiology and biochemistry. Oxygen and nitrogen saturated batch cultures of V. harveyi showed that both, IFP and oxygen consumption increased for 0.8 hours but then stopped similar to natural bacterial communities supporting the above relationship of IFP to prokaryote respiration. Our method implies adding 0.5 mM INT to a plankton sample and incubating for less than 1 hour. After prokaryote separation by size filtration (0.8 mm), the formazan crystals are collected by filtration (0.2 mm) and dissolved in propanol. The absorbance at 485 nm per sample volume yields the formazan potential that is related to prokaryote respiration in the sample.

Virus-Vectored Influenza Virus Vaccines

Science.gov (United States)

Tripp, Ralph A.; Tompkins, S. Mark

2014-01-01

Despite the availability of an inactivated vaccine that has been licensed for >50 years, the influenza virus continues to cause morbidity and mortality worldwide. Constant evolution of circulating influenza virus strains and the emergence of new strains diminishes the effectiveness of annual vaccines that rely on a match with circulating influenza strains. Thus, there is a continued need for new, efficacious vaccines conferring cross-clade protection to avoid the need for biannual reformulation of seasonal influenza vaccines. Recombinant virus-vectored vaccines are an appealing alternative to classical inactivated vaccines because virus vectors enable native expression of influenza antigens, even from virulent influenza viruses, while expressed in the context of the vector that can improve immunogenicity. In addition, a vectored vaccine often enables delivery of the vaccine to sites of inductive immunity such as the respiratory tract enabling protection from influenza virus infection. Moreover, the ability to readily manipulate virus vectors to produce novel influenza vaccines may provide the quickest path toward a universal vaccine protecting against all influenza viruses. This review will discuss experimental virus-vectored vaccines for use in humans, comparing them to licensed vaccines and the hurdles faced for licensure of these next-generation influenza virus vaccines. PMID:25105278

ProOpDB: Prokaryotic Operon DataBase.

Science.gov (United States)

Taboada, Blanca; Ciria, Ricardo; Martinez-Guerrero, Cristian E; Merino, Enrique

2012-01-01

The Prokaryotic Operon DataBase (ProOpDB, http://operons.ibt.unam.mx/OperonPredictor) constitutes one of the most precise and complete repositories of operon predictions now available. Using our novel and highly accurate operon identification algorithm, we have predicted the operon structures of more than 1200 prokaryotic genomes. ProOpDB offers diverse alternatives by which a set of operon predictions can be retrieved including: (i) organism name, (ii) metabolic pathways, as defined by the KEGG database, (iii) gene orthology, as defined by the COG database, (iv) conserved protein domains, as defined by the Pfam database, (v) reference gene and (vi) reference operon, among others. In order to limit the operon output to non-redundant organisms, ProOpDB offers an efficient method to select the most representative organisms based on a precompiled phylogenetic distances matrix. In addition, the ProOpDB operon predictions are used directly as the input data of our Gene Context Tool to visualize their genomic context and retrieve the sequence of their corresponding 5' regulatory regions, as well as the nucleotide or amino acid sequences of their genes.

Vector for IS element entrapment and functional characterization based on turning on expression of distal promoterless genes.

Science.gov (United States)

Szeverényi, I; Hodel, A; Arber, W; Olasz, F

1996-09-26

We constructed and characterized a novel trap vector for rapid isolation of insertion sequences. The strategy used for the isolation of IS elements is based on the ability of many IS elements to turn on the expression of otherwise silent genes distal to some sites of insertion. The simple transposition of an IS element can sometimes cause the constitutive expression of promoterless antibiotic resistance genes resulting in selectable phenotypes. The trap vector pAW1326 is based on a pBR322 replicon, it carries ampicillin and streptomycin resistance genes, and also silenced genes that confer chloramphenicol and kanamycin resistance once activated. The trap vector pAW1326 proved to be efficient and 85 percent of all isolated mutations were insertions. The majority of IS elements resident in the studied Escherichia coli strains tested became trapped, namely IS2, IS3, IS5, IS150, IS186 and Tn1000. We also encountered an insertion sequence, called IS10L/R-2, which is a hybrid of the two IS variants IS10L and IS10R. IS10L/R-2 is absent from most E. coli strains, but it is detectable in some strains such as JM109 which had been submitted to Tn10 mutagenesis. The distribution of the insertion sequences within the trap region was not random. Rather, the integration of chromosomal mobile genetic elements into the offered target sequence occurred in element-specific clusters. This is explained both by the target specificity and by the specific requirements for the activation of gene transcription by the DNA rearrangement. The employed trap vector pAW1326 proved to be useful for the isolation of mobile genetic elements, for a demonstration of their transposition activity as well as for the further characterization of some of the functional parameters of transposition.

Patterns of prokaryotic lateral gene transfers affecting parasitic microbial eukaryotes

DEFF Research Database (Denmark)

Alsmark, Cecilia; Foster, Peter G; Sicheritz-Pontén, Thomas

2013-01-01

BACKGROUND: The influence of lateral gene transfer on gene origins and biology in eukaryotes is poorly understood compared with those of prokaryotes. A number of independent investigations focusing on specific genes, individual genomes, or specific functional categories from various eukaryotes have...... approach to systematically investigate lateral gene transfer affecting the proteomes of thirteen, mainly parasitic, microbial eukaryotes, representing four of the six eukaryotic super-groups. All of the genomes investigated have been significantly affected by prokaryote-to-eukaryote lateral gene transfers...... indicated that lateral gene transfer does indeed affect eukaryotic genomes. However, the lack of common methodology and criteria in these studies makes it difficult to assess the general importance and influence of lateral gene transfer on eukaryotic genome evolution. RESULTS: We used a phylogenomic...

Comparative genomic analysis of translation initiation mechanisms for genes lacking the Shine–Dalgarno sequence in prokaryotes

KAUST Repository

Nakagawa, So

2017-02-15

In prokaryotes, translation initiation is believed to occur through an interaction between the 3\\' tail of a 16S rRNA and a corresponding Shine-Dalgarno (SD) sequence in the 5\\' untranslated region (UTR) of an mRNA. However, some genes lack SD sequences (non-SD genes), and the fraction of non-SD genes in a genome varies depending on the prokaryotic species. To elucidate non-SD translation initiation mechanisms in prokaryotes from an evolutionary perspective, we statistically examined the nucleotide frequencies around the initiation codons in non-SD genes from 260 prokaryotes (235 bacteria and 25 archaea). We identified distinct nucleotide frequency biases upstream of the initiation codon in bacteria and archaea, likely because of the presence of leaderless mRNAs lacking a 5\\' UTR. Moreover, we observed overall similarities in the nucleotide patterns between upstream and downstream regions of the initiation codon in all examined phyla. Symmetric nucleotide frequency biases might facilitate translation initiation by preventing the formation of secondary structures around the initiation codon. These features are more prominent in species\\' genomes that harbor large fractions of non-SD sequences, suggesting that a reduced stability around the initiation codon is important for efficient translation initiation in prokaryotes.

Comparative genomic analysis of translation initiation mechanisms for genes lacking the Shine–Dalgarno sequence in prokaryotes

KAUST Repository

Nakagawa, So; Niimura, Yoshihito; Gojobori, Takashi

2017-01-01

In prokaryotes, translation initiation is believed to occur through an interaction between the 3' tail of a 16S rRNA and a corresponding Shine-Dalgarno (SD) sequence in the 5' untranslated region (UTR) of an mRNA. However, some genes lack SD sequences (non-SD genes), and the fraction of non-SD genes in a genome varies depending on the prokaryotic species. To elucidate non-SD translation initiation mechanisms in prokaryotes from an evolutionary perspective, we statistically examined the nucleotide frequencies around the initiation codons in non-SD genes from 260 prokaryotes (235 bacteria and 25 archaea). We identified distinct nucleotide frequency biases upstream of the initiation codon in bacteria and archaea, likely because of the presence of leaderless mRNAs lacking a 5' UTR. Moreover, we observed overall similarities in the nucleotide patterns between upstream and downstream regions of the initiation codon in all examined phyla. Symmetric nucleotide frequency biases might facilitate translation initiation by preventing the formation of secondary structures around the initiation codon. These features are more prominent in species' genomes that harbor large fractions of non-SD sequences, suggesting that a reduced stability around the initiation codon is important for efficient translation initiation in prokaryotes.

Evolution of glutamate dehydrogenase genes: evidence for lateral gene transfer within and between prokaryotes and eukaryotes

Directory of Open Access Journals (Sweden)

Roger Andrew J

2003-06-01

Full Text Available Abstract Background Lateral gene transfer can introduce genes with novel functions into genomes or replace genes with functionally similar orthologs or paralogs. Here we present a study of the occurrence of the latter gene replacement phenomenon in the four gene families encoding different classes of glutamate dehydrogenase (GDH, to evaluate and compare the patterns and rates of lateral gene transfer (LGT in prokaryotes and eukaryotes. Results We extend the taxon sampling of gdh genes with nine new eukaryotic sequences and examine the phylogenetic distribution pattern of the various GDH classes in combination with maximum likelihood phylogenetic analyses. The distribution pattern analyses indicate that LGT has played a significant role in the evolution of the four gdh gene families. Indeed, a number of gene transfer events are identified by phylogenetic analyses, including numerous prokaryotic intra-domain transfers, some prokaryotic inter-domain transfers and several inter-domain transfers between prokaryotes and microbial eukaryotes (protists. Conclusion LGT has apparently affected eukaryotes and prokaryotes to a similar extent within the gdh gene families. In the absence of indications that the evolution of the gdh gene families is radically different from other families, these results suggest that gene transfer might be an important evolutionary mechanism in microbial eukaryote genome evolution.

Effects of Predation by Protists on Prokaryotic Community Function, Structure, and Diversity in Anaerobic Granular Sludge.

Science.gov (United States)

Hirakata, Yuga; Oshiki, Mamoru; Kuroda, Kyohei; Hatamoto, Masashi; Kubota, Kengo; Yamaguchi, Takashi; Harada, Hideki; Araki, Nobuo

2016-09-29

Predation by protists is top-down pressure that regulates prokaryotic abundance, community function, structure, and diversity in natural and artificial ecosystems. Although the effects of predation by protists have been studied in aerobic ecosystems, they are poorly understood in anoxic environments. We herein studied the influence of predation by Metopus and Caenomorpha ciliates-ciliates frequently found in anoxic ecosystems-on prokaryotic community function, structure, and diversity. Metopus and Caenomorpha ciliates were cocultivated with prokaryotic assemblages (i.e., anaerobic granular sludge) in an up-flow anaerobic sludge blanket (UASB) reactor for 171 d. Predation by these ciliates increased the methanogenic activities of granular sludge, which constituted 155% of those found in a UASB reactor without the ciliates (i.e., control reactor). Sequencing of 16S rRNA gene amplicons using Illumina MiSeq revealed that the prokaryotic community in the UASB reactor with the ciliates was more diverse than that in the control reactor; 2,885-3,190 and 2,387-2,426 operational taxonomic units (>97% sequence similarities), respectively. The effects of predation by protists in anaerobic engineered systems have mostly been overlooked, and our results show that the influence of predation by protists needs to be examined and considered in the future for a better understanding of prokaryotic community structure and function.

Influenza viral vectors expressing the Brucella OMP16 or L7/L12 proteins as vaccines against B. abortus infection.

Science.gov (United States)

Tabynov, Kaissar; Sansyzbay, Abylai; Kydyrbayev, Zhailaubay; Yespembetov, Bolat; Ryskeldinova, Sholpan; Zinina, Nadezhda; Assanzhanova, Nurika; Sultankulova, Kulaisan; Sandybayev, Nurlan; Khairullin, Berik; Kuznetsova, Irina; Ferko, Boris; Egorov, Andrej

2014-04-10

We generated novel, effective candidate vaccine against Brucella abortus based on recombinant influenza viruses expressing the Brucella ribosomal protein L7/L12 or outer membrane protein (Omp)-16 from the NS1 open reading frame. The main purpose of this work was to evaluate the safety, immunogenicity and protectiveness of vaccine candidate in laboratory animals. Four recombinant influenza A viral constructs of the subtypes Н5N1 or H1N1 expressing the Brucella proteins L7/L12 or Omp16 were obtained by a reverse genetics method: Flu-NS1-124-L7/L12-H5N1, Flu-NS1-124-Omp16-H5N1, Flu-NS1-124-L7/L12-H1N1 and Flu-NS1-124-Omp16-H1N1. Despite of substantial modification of NS1 gene, all constructs replicated well and were retain their Brucella inserts over five passages in embryonated chicken eggs (CE). Administration of the mono- or bivalent vaccine formulation via prime-boost intranasal (i.n.), conjunctival (c.) or subcutaneous (s.c.) immunization was safe in mice; no deaths, body weight loss or pathomorphological changes were observed over 56 days. Moreover, guinea pigs vaccinated i.n. with vaccine vectors did not shed the vaccine viruses through their upper respiratory tract after the prime and booster vaccination. These findings confirmed the replication-deficient phenotype of viral vectors. The highest antibody response to Brucella antigen was obtained with constructs expressing L7/L12 (ELISA, GMT 242.5-735.0); whereas the highest T-cell immune response- with construct expressing Omp16 (ELISPOT, 337 ± 52-651 ± 45 spots/4×105cells), which was comparable (P > 0.05) to the response induced by the commercial vaccine B. abortus 19. Interestingly, c. immunization appeared to be optimal for eliciting T-cell immune response. In guinea pigs, the highest protective efficacy after challenge with B. abortus 544 was achieved with Omp16 expressing constructs in both monovalent or bivalent vaccine formulations; protective efficacy was comparable to those induced by

Prokaryotic communities in pit mud from different-aged cellars used for the production of Chinese strong-flavored liquor.

Science.gov (United States)

Tao, Yong; Li, Jiabao; Rui, Junpeng; Xu, Zhancheng; Zhou, Yan; Hu, Xiaohong; Wang, Xiang; Liu, Menghua; Li, Daping; Li, Xiangzhen

2014-04-01

Chinese strong-flavored liquor (CSFL) accounts for more than 70% of all Chinese liquor production. Microbes in pit mud play key roles in the fermentation cellar for the CSFL production. However, microbial diversity, community structure, and cellar-age-related changes in pit mud are poorly understood. Here, we investigated the prokaryotic community structure and diversity in pit-mud samples with different cellar ages (1, 10, 25, and 50 years) using the pyrosequencing technique. Results indicated that prokaryotic diversity increased with cellar age until the age reached 25 years and that prokaryotic community structure changed significantly between three cellar ages (1, 10, and 25 years). Significant correlations between prokaryotic communities and environmental variables (pH, NH4(+), lactic acid, butyric acid, and caproic acid) were observed. Overall, our study results suggested that the long-term brewing operation shapes unique prokaryotic community structure and diversity as well as pit-mud chemistry. We have proposed a three-phase model to characterize the changes of pit-mud prokaryotic communities. (i) Phase I is an initial domestication period. Pit mud is characterized by abundant Lactobacillus and high lactic acid and low pH levels. (ii) Phase II is a transition period. While Lactobacillus abundance decreases dramatically, that of Bacteroidetes and methanogens increases. (iii) Phase III is a relative mature period. The prokaryotic community shows the highest diversity and capability to produce more caproic acid as a precursor for synthesis of ethyl caproate, the main flavor component in CSFL. This research provides scientific evidence to support the practical experience that old fermentation cellars produce high-quality liquor.

Read length and repeat resolution: Exploring prokaryote genomes using next-generation sequencing technologies

KAUST Repository

Cahill, Matt J.

2010-07-12

Background: There are a growing number of next-generation sequencing technologies. At present, the most cost-effective options also produce the shortest reads. However, even for prokaryotes, there is uncertainty concerning the utility of these technologies for the de novo assembly of complete genomes. This reflects an expectation that short reads will be unable to resolve small, but presumably abundant, repeats. Methodology/Principal Findings: Using a simple model of repeat assembly, we develop and test a technique that, for any read length, can estimate the occurrence of unresolvable repeats in a genome, and thus predict the number of gaps that would need to be closed to produce a complete sequence. We apply this technique to 818 prokaryote genome sequences. This provides a quantitative assessment of the relative performance of various lengths. Notably, unpaired reads of only 150nt can reconstruct approximately 50% of the analysed genomes with fewer than 96 repeat-induced gaps. Nonetheless, there is considerable variation amongst prokaryotes. Some genomes can be assembled to near contiguity using very short reads while others require much longer reads. Conclusions: Given the diversity of prokaryote genomes, a sequencing strategy should be tailored to the organism under study. Our results will provide researchers with a practical resource to guide the selection of the appropriate read length. 2010 Cahill et al.

Read length and repeat resolution: exploring prokaryote genomes using next-generation sequencing technologies.

Directory of Open Access Journals (Sweden)

Matt J Cahill

Full Text Available BACKGROUND: There are a growing number of next-generation sequencing technologies. At present, the most cost-effective options also produce the shortest reads. However, even for prokaryotes, there is uncertainty concerning the utility of these technologies for the de novo assembly of complete genomes. This reflects an expectation that short reads will be unable to resolve small, but presumably abundant, repeats. METHODOLOGY/PRINCIPAL FINDINGS: Using a simple model of repeat assembly, we develop and test a technique that, for any read length, can estimate the occurrence of unresolvable repeats in a genome, and thus predict the number of gaps that would need to be closed to produce a complete sequence. We apply this technique to 818 prokaryote genome sequences. This provides a quantitative assessment of the relative performance of various lengths. Notably, unpaired reads of only 150nt can reconstruct approximately 50% of the analysed genomes with fewer than 96 repeat-induced gaps. Nonetheless, there is considerable variation amongst prokaryotes. Some genomes can be assembled to near contiguity using very short reads while others require much longer reads. CONCLUSIONS: Given the diversity of prokaryote genomes, a sequencing strategy should be tailored to the organism under study. Our results will provide researchers with a practical resource to guide the selection of the appropriate read length.

Read length and repeat resolution: Exploring prokaryote genomes using next-generation sequencing technologies

KAUST Repository

Cahill, Matt J.; Kö ser, Claudio U.; Ross, Nicholas E.; Archer, John A.C.

2010-01-01

Background: There are a growing number of next-generation sequencing technologies. At present, the most cost-effective options also produce the shortest reads. However, even for prokaryotes, there is uncertainty concerning the utility of these technologies for the de novo assembly of complete genomes. This reflects an expectation that short reads will be unable to resolve small, but presumably abundant, repeats. Methodology/Principal Findings: Using a simple model of repeat assembly, we develop and test a technique that, for any read length, can estimate the occurrence of unresolvable repeats in a genome, and thus predict the number of gaps that would need to be closed to produce a complete sequence. We apply this technique to 818 prokaryote genome sequences. This provides a quantitative assessment of the relative performance of various lengths. Notably, unpaired reads of only 150nt can reconstruct approximately 50% of the analysed genomes with fewer than 96 repeat-induced gaps. Nonetheless, there is considerable variation amongst prokaryotes. Some genomes can be assembled to near contiguity using very short reads while others require much longer reads. Conclusions: Given the diversity of prokaryote genomes, a sequencing strategy should be tailored to the organism under study. Our results will provide researchers with a practical resource to guide the selection of the appropriate read length. 2010 Cahill et al.

A stable RNA virus-based vector for citrus trees

International Nuclear Information System (INIS)

Folimonov, Alexey S.; Folimonova, Svetlana Y.; Bar-Joseph, Moshe; Dawson, William O.

2007-01-01

Virus-based vectors are important tools in plant molecular biology and plant genomics. A number of vectors based on viruses that infect herbaceous plants are in use for expression or silencing of genes in plants as well as screening unknown sequences for function. Yet there is a need for useful virus-based vectors for woody plants, which demand much greater stability because of the longer time required for systemic infection and analysis. We examined several strategies to develop a Citrus tristeza virus (CTV)-based vector for transient expression of foreign genes in citrus trees using a green fluorescent protein (GFP) as a reporter. These strategies included substitution of the p13 open reading frame (ORF) by the ORF of GFP, construction of a self-processing fusion of GFP in-frame with the major coat protein (CP), or expression of the GFP ORF as an extra gene from a subgenomic (sg) mRNA controlled either by a duplicated CTV CP sgRNA controller element (CE) or an introduced heterologous CE of Beet yellows virus. Engineered vector constructs were examined for replication, encapsidation, GFP expression during multiple passages in protoplasts, and for their ability to infect, move, express GFP, and be maintained in citrus plants. The most successful vectors based on the 'add-a-gene' strategy have been unusually stable, continuing to produce GFP fluorescence after more than 4 years in citrus trees

Food-grade host/vector expression system for Lactobacillus casei based on complementation of plasmid-associated phospho-beta-galactosidase gene lacG.

Science.gov (United States)

Takala, T M; Saris, P E J; Tynkkynen, S S H

2003-01-01

A new food-grade host/vector system for Lactobacillus casei based on lactose selection was constructed. The wild-type non-starter host Lb. casei strain E utilizes lactose via a plasmid-encoded phosphotransferase system. For food-grade cloning, a stable lactose-deficient mutant was constructed by deleting a 141-bp fragment from the phospho-beta-galactosidase gene lacG via gene replacement. The deletion resulted in an inactive phospho-beta-galactosidase enzyme with an internal in-frame deletion of 47 amino acids. A complementation plasmid was constructed containing a replicon from Lactococcus lactis, the lacG gene from Lb. casei, and the constitutive promoter of pepR for lacG expression from Lb. rhamnosus. The expression of the lacG gene from the resulting food-grade plasmid pLEB600 restored the ability of the lactose-negative mutant strain to grow on lactose to the wild-type level. The vector pLEB600 was used for expression of the proline iminopeptidase gene pepI from Lb. helveticus in Lb. casei. The results show that the food-grade expression system reported in this paper can be used for expression of foreign genes in Lb. casei.

BLAST Ring Image Generator (BRIG: simple prokaryote genome comparisons

Directory of Open Access Journals (Sweden)

Beatson Scott A

2011-08-01

Full Text Available Abstract Background Visualisation of genome comparisons is invaluable for helping to determine genotypic differences between closely related prokaryotes. New visualisation and abstraction methods are required in order to improve the validation, interpretation and communication of genome sequence information; especially with the increasing amount of data arising from next-generation sequencing projects. Visualising a prokaryote genome as a circular image has become a powerful means of displaying informative comparisons of one genome to a number of others. Several programs, imaging libraries and internet resources already exist for this purpose, however, most are either limited in the number of comparisons they can show, are unable to adequately utilise draft genome sequence data, or require a knowledge of command-line scripting for implementation. Currently, there is no freely available desktop application that enables users to rapidly visualise comparisons between hundreds of draft or complete genomes in a single image. Results BLAST Ring Image Generator (BRIG can generate images that show multiple prokaryote genome comparisons, without an arbitrary limit on the number of genomes compared. The output image shows similarity between a central reference sequence and other sequences as a set of concentric rings, where BLAST matches are coloured on a sliding scale indicating a defined percentage identity. Images can also include draft genome assembly information to show read coverage, assembly breakpoints and collapsed repeats. In addition, BRIG supports the mapping of unassembled sequencing reads against one or more central reference sequences. Many types of custom data and annotations can be shown using BRIG, making it a versatile approach for visualising a range of genomic comparison data. BRIG is readily accessible to any user, as it assumes no specialist computational knowledge and will perform all required file parsing and BLAST comparisons

BLAST Ring Image Generator (BRIG): simple prokaryote genome comparisons.

Science.gov (United States)

Alikhan, Nabil-Fareed; Petty, Nicola K; Ben Zakour, Nouri L; Beatson, Scott A

2011-08-08

Visualisation of genome comparisons is invaluable for helping to determine genotypic differences between closely related prokaryotes. New visualisation and abstraction methods are required in order to improve the validation, interpretation and communication of genome sequence information; especially with the increasing amount of data arising from next-generation sequencing projects. Visualising a prokaryote genome as a circular image has become a powerful means of displaying informative comparisons of one genome to a number of others. Several programs, imaging libraries and internet resources already exist for this purpose, however, most are either limited in the number of comparisons they can show, are unable to adequately utilise draft genome sequence data, or require a knowledge of command-line scripting for implementation. Currently, there is no freely available desktop application that enables users to rapidly visualise comparisons between hundreds of draft or complete genomes in a single image. BLAST Ring Image Generator (BRIG) can generate images that show multiple prokaryote genome comparisons, without an arbitrary limit on the number of genomes compared. The output image shows similarity between a central reference sequence and other sequences as a set of concentric rings, where BLAST matches are coloured on a sliding scale indicating a defined percentage identity. Images can also include draft genome assembly information to show read coverage, assembly breakpoints and collapsed repeats. In addition, BRIG supports the mapping of unassembled sequencing reads against one or more central reference sequences. Many types of custom data and annotations can be shown using BRIG, making it a versatile approach for visualising a range of genomic comparison data. BRIG is readily accessible to any user, as it assumes no specialist computational knowledge and will perform all required file parsing and BLAST comparisons automatically. There is a clear need for a user

Protein Based Molecular Markers Provide Reliable Means to Understand Prokaryotic Phylogeny and Support Darwinian Mode of Evolution

Directory of Open Access Journals (Sweden)

Vaibhav eBhandari

2012-07-01

Full Text Available The analyses of genome sequences have led to the proposal that lateral gene transfers (LGTs among prokaryotes are so widespread that they disguise the interrelationships among these organisms. This has led to questioning whether the Darwinian model of evolution is applicable to the prokaryotic organisms. In this review, we discuss the usefulness of taxon-specific molecular markers such as conserved signature indels (CSIs and conserved signature proteins (CSPs for understanding the evolutionary relationships among prokaryotes and to assess the influence of LGTs on prokaryotic evolution. The analyses of genomic sequences have identified large numbers of CSIs and CSPs that are unique properties of different groups of prokaryotes ranging from phylum to genus levels. The species distribution patterns of these molecular signatures strongly support a tree-like vertical inheritance of the genes containing these molecular signatures that is consistent with phylogenetic trees. Recent detailed studies in this regard on Thermotogae and Archaea, which are reviewed here, have identified large numbers of CSIs and CSPs that are specific for the species from these two taxa and a number of their major clades. The genetic changes responsible for these CSIs (and CSPs initially likely occurred in the common ancestors of these taxa and then vertically transferred to various descendants. Although some CSIs and CSPs in unrelated groups of prokaryotes were identified, their small numbers and random occurrence has no apparent influence on the consistent tree-like branching pattern emerging from other markers. These results provide evidence that although LGT is an important evolutionary force, it does not mask the tree-like branching pattern of prokaryotes or understanding of their evolutionary relationships. The identified CSIs and CSPs also provide novel and highly specific means for identification of different groups of microbes and for taxonomical and biochemical

Protein based molecular markers provide reliable means to understand prokaryotic phylogeny and support Darwinian mode of evolution.

Science.gov (United States)

Bhandari, Vaibhav; Naushad, Hafiz S; Gupta, Radhey S

2012-01-01

The analyses of genome sequences have led to the proposal that lateral gene transfers (LGTs) among prokaryotes are so widespread that they disguise the interrelationships among these organisms. This has led to questioning of whether the Darwinian model of evolution is applicable to prokaryotic organisms. In this review, we discuss the usefulness of taxon-specific molecular markers such as conserved signature indels (CSIs) and conserved signature proteins (CSPs) for understanding the evolutionary relationships among prokaryotes and to assess the influence of LGTs on prokaryotic evolution. The analyses of genomic sequences have identified large numbers of CSIs and CSPs that are unique properties of different groups of prokaryotes ranging from phylum to genus levels. The species distribution patterns of these molecular signatures strongly support a tree-like vertical inheritance of the genes containing these molecular signatures that is consistent with phylogenetic trees. Recent detailed studies in this regard on the Thermotogae and Archaea, which are reviewed here, have identified large numbers of CSIs and CSPs that are specific for the species from these two taxa and a number of their major clades. The genetic changes responsible for these CSIs (and CSPs) initially likely occurred in the common ancestors of these taxa and then vertically transferred to various descendants. Although some CSIs and CSPs in unrelated groups of prokaryotes were identified, their small numbers and random occurrence has no apparent influence on the consistent tree-like branching pattern emerging from other markers. These results provide evidence that although LGT is an important evolutionary force, it does not mask the tree-like branching pattern of prokaryotes or understanding of their evolutionary relationships. The identified CSIs and CSPs also provide novel and highly specific means for identification of different groups of microbes and for taxonomical and biochemical studies.

A quantitative account of genomic island acquisitions in prokaryotes

Directory of Open Access Journals (Sweden)

Roos Tom E

2011-08-01

Full Text Available Abstract Background Microbial genomes do not merely evolve through the slow accumulation of mutations, but also, and often more dramatically, by taking up new DNA in a process called horizontal gene transfer. These innovation leaps in the acquisition of new traits can take place via the introgression of single genes, but also through the acquisition of large gene clusters, which are termed Genomic Islands. Since only a small proportion of all the DNA diversity has been sequenced, it can be hard to find the appropriate donors for acquired genes via sequence alignments from databases. In contrast, relative oligonucleotide frequencies represent a remarkably stable genomic signature in prokaryotes, which facilitates compositional comparisons as an alignment-free alternative for phylogenetic relatedness. In this project, we test whether Genomic Islands identified in individual bacterial genomes have a similar genomic signature, in terms of relative dinucleotide frequencies, and can therefore be expected to originate from a common donor species. Results When multiple Genomic Islands are present within a single genome, we find that up to 28% of these are compositionally very similar to each other, indicative of frequent recurring acquisitions from the same donor to the same acceptor. Conclusions This represents the first quantitative assessment of common directional transfer events in prokaryotic evolutionary history. We suggest that many of the resident Genomic Islands per prokaryotic genome originated from the same source, which may have implications with respect to their regulatory interactions, and for the elucidation of the common origins of these acquired gene clusters.

Inference of sigma factor controlled networks by using numerical modeling applied to microarray time series data of the germinating prokaryote.

Science.gov (United States)

Strakova, Eva; Zikova, Alice; Vohradsky, Jiri

2014-01-01

A computational model of gene expression was applied to a novel test set of microarray time series measurements to reveal regulatory interactions between transcriptional regulators represented by 45 sigma factors and the genes expressed during germination of a prokaryote Streptomyces coelicolor. Using microarrays, the first 5.5 h of the process was recorded in 13 time points, which provided a database of gene expression time series on genome-wide scale. The computational modeling of the kinetic relations between the sigma factors, individual genes and genes clustered according to the similarity of their expression kinetics identified kinetically plausible sigma factor-controlled networks. Using genome sequence annotations, functional groups of genes that were predominantly controlled by specific sigma factors were identified. Using external binding data complementing the modeling approach, specific genes involved in the control of the studied process were identified and their function suggested.

Exploring cultivable Bacteria from the prokaryotic community associated with the carnivorous sponge Asbestopluma hypogea.

Science.gov (United States)

Dupont, Samuel; Carre-Mlouka, Alyssa; Domart-Coulon, Isabelle; Vacelet, Jean; Bourguet-Kondracki, Marie-Lise

2014-04-01

Combining culture-dependent and independent approaches, we investigated for the first time the cultivable fraction of the prokaryotic community associated with the carnivorous sponge Asbestopluma hypogea. The heterotrophic prokaryotes isolated from this tiny sponge were compared between specimens freshly collected from cave and maintained in aquarium. Overall, 67 isolates obtained in pure culture were phylogenetically affiliated to the bacterial phyla Proteobacteria, Bacteroidetes, Actinobacteria, and Firmicutes. This cultivable diversity was lower than the prokaryotic diversity obtained by previous pyrosequencing study and comparable to that of another Mediterranean demosponge, the filter-feeding Phorbas tenacior. Furthermore, using fluorescence in situ hybridization, we visualized bacterial and archaeal cells, confirming the presence of both prokaryotes in A. hypogea tissue. Approximately 16% of the bacterial isolates tested positive for chitinolytic activity, suggesting potential microbial involvement in the digestion processes of crustacean prey by this carnivorous sponge. Additionally, 6% and 16% of bacterial isolates revealed antimicrobial and antioxidant activities, respectively. One Streptomyces sp. S1CA strain was identified as a promising candidate for the production of antimicrobial and antioxidant secondary metabolites as well as chitinolytic enzymes. Implications in the context of the sponge biology and prey-feeding strategy are discussed. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

DNA sequence and prokaryotic expression analysis of vitellogenin ...

African Journals Online (AJOL)

In this study, the DNA sequence of vitellogenin from Antheraea pernyi (Ap-Vg) was identified and its functional domain (30-740 aa, Ap-Vg-1) was expressed in Escherichia coli BL21 (DE3) cells. The recombinant Ap-Vg-1 proteins were purified and used for antibody preparation. The results showed that the intact DNA ...

Effects of Predation by Protists on Prokaryotic Community Function, Structure, and Diversity in Anaerobic Granular Sludge

Science.gov (United States)

Hirakata, Yuga; Oshiki, Mamoru; Kuroda, Kyohei; Hatamoto, Masashi; Kubota, Kengo; Yamaguchi, Takashi; Harada, Hideki; Araki, Nobuo

2016-01-01

Predation by protists is top-down pressure that regulates prokaryotic abundance, community function, structure, and diversity in natural and artificial ecosystems. Although the effects of predation by protists have been studied in aerobic ecosystems, they are poorly understood in anoxic environments. We herein studied the influence of predation by Metopus and Caenomorpha ciliates—ciliates frequently found in anoxic ecosystems—on prokaryotic community function, structure, and diversity. Metopus and Caenomorpha ciliates were cocultivated with prokaryotic assemblages (i.e., anaerobic granular sludge) in an up-flow anaerobic sludge blanket (UASB) reactor for 171 d. Predation by these ciliates increased the methanogenic activities of granular sludge, which constituted 155% of those found in a UASB reactor without the ciliates (i.e., control reactor). Sequencing of 16S rRNA gene amplicons using Illumina MiSeq revealed that the prokaryotic community in the UASB reactor with the ciliates was more diverse than that in the control reactor; 2,885–3,190 and 2,387–2,426 operational taxonomic units (>97% sequence similarities), respectively. The effects of predation by protists in anaerobic engineered systems have mostly been overlooked, and our results show that the influence of predation by protists needs to be examined and considered in the future for a better understanding of prokaryotic community structure and function. PMID:27431197

A Novel Prokaryotic Green Fluorescent Protein Expression System for Testing Gene Editing Tools Activity Like Zinc Finger Nuclease.

Science.gov (United States)

Sabzehei, Faezeh; Kouhpayeh, Shirin; Dastjerdeh, Mansoureh Shahbazi; Khanahmad, Hossein; Salehi, Rasoul; Naderi, Shamsi; Taghizadeh, Razieh; Rabiei, Parisa; Hejazi, Zahra; Shariati, Laleh

2017-01-01

Gene editing technology has created a revolution in the field of genome editing. The three of the most famous tools in gene editing technology are zinc finger nucleases (ZFNs), transcription activator-like effector nucleases, clustered regularly interspaced short palindromic repeats (CRISPR), and CRISPR-associated systems. As their predictable nature, it is necessary to assess their efficiency. There are some methods for this purpose, but most of them are time labor and complicated. Here, we introduce a new prokaryotic reporter system, which makes it possible to evaluate the efficiency of gene editing tools faster, cheaper, and simpler than previous methods. At first, the target sites of a custom ZFN, which is designed against a segment of ampicillin resistance gene, were cloned on both sides of green fluorescent protein (GFP) gene to construct pPRO-GFP. Then pPRO-GFP was transformed into Escherichia coli TOP10F' that contains pZFN (contains expression cassette of a ZFN against ampicillin resistant gene), or p15A-KanaR as a negative control. The transformed bacteria were cultured on three separate media that contained ampicillin, kanamycin, and ampicillin + kanamycin; then the resulted colonies were assessed by flow cytometry. The results of flow cytometry showed a significant difference between the case (bacteria contain pZFN) and control (bacteria contain p15A, KanaR) in MFI (Mean Fluorescence Intensity) ( P < 0.0001). According to ZFN efficiency, it can bind and cut the target sites, the bilateral cutting can affect the intensity of GFP fluorescence. Our flow cytometry results showed that this ZFN could reduce the intensity of GFP color and colony count of bacteria in media containing amp + kana versus control sample.

Temperature regulation of marine heterotrophic prokaryotes increases latitudinally as a breach between bottom-up and top-down controls

KAUST Repository

Moran, Xose Anxelu G.; Gasol, Josep M.; Pernice, Massimo C.; Mangot, Jean-Franç ois; Massana, Ramon; Lara, Elena; Vaqué , Dolors; Duarte, Carlos M.

2017-01-01

Planktonic heterotrophic prokaryotes make up the largest living biomass and process most organic matter in the ocean. Determining when and where the biomass and activity of heterotrophic prokaryotes are controlled by resource availability (bottom

Construction and identification of double-gene co-expression vector with radiation-inducible human TRAIL and endostatin

International Nuclear Information System (INIS)

Li Yanbo; Guo Caixia; Gong Pingsheng; Liu Yang; Liangshuo; Wang Hongfang; Wang Jianfeng; Gong Shouliang

2010-01-01

Objective: To construct a recombinant plasmid pshuttle-Egr1-shTRAIL-shES containing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and endostatin double genes. Methods: The secretary endostatin gene (shES) fragment was amplified from the pMD19T-endostatin vector by PCR. The shES gene was ligated to pMD19Tand sequenced. Finally, using the gene recombinant technique, the recombinant plasmid pshuttle-Egr1- shTRAIL-shES with radiation-inducible Egr1 promoter, secretary TRAIL and endostatin double-gene was constructed. Results: The sequence of the shES gene was in concordance with that anticipated indicating shES gene was acquired successfully.Moreover, the results acquired by PCR and restrictive digestion identification of the recombinant plasmid pshuttle-Egr1-shTRAIL-shES and all the vectors refered to its construction confirmed that pshuttle-Egr1-shTRAIL-shES was constructed correctly. Conclusion: The radiation-inducible double-gene co-expression vector pshuttle-Egr1-shTRAIL-shES is constructed successfully, which would set the experimental foundation for further study on the anti-tumor effect of TRAIL and endostatin double-gene-radiotherapy and its related mechanisms. (authors)

Construction of Egr1-mediated human truncated apoptosis inducing factor expression vector and its expression regularity induced by radiation in breast cancer MCF-7 cells

International Nuclear Information System (INIS)

Wang Jianfeng; Gong Shouliang; Wang Zhicheng; Fang Fang; Liu Yang; Wu Jiahui

2012-01-01

Objective: To clone human truncated apoptosis inducing factor (AIF) cDNA sequence, and to construct early growth response 1 (Egr1)-mediated recombinant expression vector pcDNA 3.1-Egr1-AIF Δ1-480 (pEgr1-AIFΔ 1-480 ), and to observe its regularity induced by radiation in human breast cancer MCF-7 cells. Methods: The total mRNA extracted from human leukemia Jurkat cells used as template, and the human AIFΔ 1-480 was acquired by RT-PCR, and it was linked to pMD18T vector for sequencing. Egr1 fragment was digested from pMD19T-Egr1 by restrictive enzyme, and the Egr1-mediated expression plasmid pEgr1-AIFΔ 1-480 was constructed by gene recombination. There were control group, pcDNA3.1 group, pAIFΔ 1-480 group and pEgr1-AIFΔ 1-480 group in the experiment. After the plasmids in various groups were transfected into human breast cancer MCF-7 cells, the AIF and AIFΔ 1-480 protein expression time-effect (0, 2, 4, 12, 24 and 48 h after 2.0 Gy irradiation) and dose-effect (24 h after 0, 0.2, 0.5, 1.0, 2.0 and 5.0 Gy irradiation) regularity were measured by Western blotting method. Results: The sequencing results showed that the AIFΔ 1-480 acquired by RT-PCR was consistent with the sequence expected, the pEgr-AIFΔ 1-480 was confirmed by PCR and restrictive enzyme digestion. After 0-48 h the MCF-7 cells were irradiated by 2.0 Gy, and the AIF protein expressed in the cells in each group, and it increased significantly from 4 h and the AIF expressions in 4, 12, 24 and 48 h groups were higher than that in 0 h group (P<0.05), and it reached to maximum value at 48 h. But the AIFΔ 1-480 protein expressed in the cells in pAIFΔ 1-480 and pEgr1-AIFΔ 1-480 groups from 2 h (P<0.05), and it reached to peak value at 24 h. The AIFΔ 1-480 expressions in pEgr1-AIFΔ 1-480 group were higher than those in pAIFΔ 1-480 group at and 48 h (P<0.05). After the MCF-7 cells were irradiated by 0-5 Gy for 24 h, the AIF protein expressed in the cells in each group, but the AIFΔ 1-480 protein

Spatiotemporal and species variations in prokaryotic communities associated with sediments from surface-flow constructed wetlands for treating swine wastewater.

Science.gov (United States)

Jia, Fen; Lai, Cui; Chen, Liang; Zeng, Guangming; Huang, Danlian; Liu, Feng; Li, Xi; Luo, Pei; Wu, Jinshui; Qin, Lei; Zhang, Chen; Cheng, Min; Xu, Piao

2017-10-01

Microorganisms are the main mechanisms of pollutants removals in constructed wetlands (CWs) used for wastewater treatment. However, the different biological processes and variations of prokaryotic community in CWs remain poorly understood. In this study, we applied a high-throughput sequencing technique to investigate the prokaryotic communities associated with sediments from pilot-scale surface-flow constructed wetlands (SFCWs) treating swine wastewater (SW) of varying strengths. Our results revealed that highly diverse prokaryotic communities were present in the SFCWs, with Proteobacteria (16.44-44.44%), Acidobacteria (3.25-24.40%), and Chloroflexi (5.77-14.43%) being the major phyla, and Nitrospira (4.14-12.02%), the most dominant genus. The prokaryotic communities in the sediments varied greatly with location and season, which markedly altered the microenvironmental conditions. Principal co-ordinates analysis indicated that SW strength significantly influenced the community structure in sediments of the SFCWs, and canonical correspondence analysis illustrated that the shifts in prokaryotic communities were strongly related to NO 3 - -N and TN in winter; and in summer with NH 4 + N, NO 3 - -N, NO 2 - -N, TN, TP, SOM, and pH. In conclusion, the use of high-throughput sequencing greatly enhanced our understanding of prokaryotic communities with different functional groups in SFCWs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Coxsackievirus B3 vaccines: use as an expression vector for prevention of myocarditis.

Science.gov (United States)

Henke, Andreas; Jarasch, Nadine; Wutzler, Peter

2008-12-01

Coxsackievirus B3 (CVB3), a member of the Picornaviridae family, is considered to be one of the most important infectious agents to cause virus-induced myocarditis. Despite improvements in studying virus pathology, structure and molecular biology, as well as the diagnosis of this disease, there is still no virus-specific drug or vaccine in clinical use. During the last 20 years many investigations have been performed to develop classic and modern immunization techniques against CVB3-induced heart disease. One promising approach among others includes the insertion of coding sequences of cytokines into the viral genome. The application of an IFN-gamma-expressing recombinant coxsackievirus vector is especially efficient against CVB3-induced myocarditis. Beside direct IFN-gamma-mediated antiviral effects, the local and simultaneous expression of IFN-gamma by the virus itself activates the immune system in a strong and long-lasting manner, which protects animals completely against subsequent lethal infections independently of the age of the immunized individual and the route of vaccine administration.

Simian virus 40 vectors for pulmonary gene therapy

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Oppenheim Ariella

2007-10-01

Full Text Available Abstract Background Sepsis remains the leading cause of death in critically ill patients. One of the primary organs affected by sepsis is the lung, presenting as the Acute Respiratory Distress Syndrome (ARDS. Organ damage in sepsis involves an alteration in gene expression, making gene transfer a potential therapeutic modality. This work examines the feasibility of applying simian virus 40 (SV40 vectors for pulmonary gene therapy. Methods Sepsis-induced ARDS was established by cecal ligation double puncture (2CLP. SV40 vectors carrying the luciferase reporter gene (SV/luc were administered intratracheally immediately after sepsis induction. Sham operated (SO as well as 2CLP rats given intratracheal PBS or adenovirus expressing luciferase served as controls. Luc transduction was evaluated by in vivo light detection, immunoassay and luciferase mRNA detection by RT-PCR in tissue harvested from septic rats. Vector abundance and distribution into alveolar cells was evaluated using immunostaining for the SV40 VP1 capsid protein as well as by double staining for VP1 and for the surfactant protein C (proSP-C. Immunostaining for T-lymphocytes was used to evaluate the cellular immune response induced by the vector. Results Luc expression measured by in vivo light detection correlated with immunoassay from lung tissue harvested from the same rats. Moreover, our results showed vector presence in type II alveolar cells. The vector did not induce significant cellular immune response. Conclusion In the present study we have demonstrated efficient uptake and expression of an SV40 vector in the lungs of animals with sepsis-induced ARDS. These vectors appear to be capable of in vivo transduction of alveolar type II cells and may thus become a future therapeutic tool.

Vector Development for the Expression of Foreign Proteins in the Vaccine Strain Brucella abortus S19

Science.gov (United States)

Comerci, Diego J.; Pollevick, Guido D.; Vigliocco, Ana M.; Frasch, Alberto C. C.; Ugalde, Rodolfo A.

1998-01-01

A vector for the expression of foreign antigens in the vaccine strain Brucella abortus S19 was developed by using a DNA fragment containing the regulatory sequences and the signal peptide of the Brucella bcsp31 gene. This fragment was cloned in broad-host-range plasmid pBBR4MCS, resulting in plasmid pBEV. As a reporter protein, a repetitive antigen of Trypanosoma cruzi was used. The recombinant fusion protein is stably expressed and secreted into the Brucella periplasmic space, inducing a good antibody response against the T. cruzi antigen. The expression of the repetitive antigen in Brucella neither altered its growth pattern nor generated a toxic or lethal effect during experimental infection. The application of this strategy for the generation of live recombinant vaccines and the tagging of B. abortus S19 vaccine is discussed. This is the first time that a recombinant protein has been expressed in the periplasm of brucellae. PMID:9673273

Mitochondrial intermediate peptidase: Expression in Escherichia coli and improvement of its enzymatic activity detection with FRET substrates

International Nuclear Information System (INIS)

Marcondes, Marcelo F.; Torquato, Ricardo J.S.; Assis, Diego M.; Juliano, Maria A.; Hayashi, Mirian A.F.; Oliveira, Vitor

2010-01-01

In the present study, soluble, functionally-active, recombinant human mitochondrial intermediate peptidase (hMIP), a mitochondrial metalloendoprotease, was expressed in a prokaryotic system. The hMIP fusion protein, with a poly-His-tag (6x His), was obtained by cloning the coding region of hMIP cDNA into the pET-28a expression vector, which was then used to transform Escherichia coli BL21 (DE3) pLysS. After isolation and purification of the fusion protein by affinity chromatography using Ni-Sepharose resin, the protein was purified further using ion exchange chromatography with a Hi-trap resource Q column. The recombinant hMIP was characterized by Western blotting using three distinct antibodies, circular dichroism, and enzymatic assays that used the first FRET substrates developed for MIP and a series of protease inhibitors. The successful expression of enzymatically-active hMIP in addition to the FRET substrates will contribute greatly to the determination of substrate specificity of this protease and to the development of specific inhibitors that are essential for a better understanding of the role of this protease in mitochondrial functioning.

Thermophilic Adaptation in Prokaryotes Is Constrained by Metabolic Costs of Proteostasis

Science.gov (United States)

Venev, Sergey V; Zeldovich, Konstantin B

2018-01-01

Abstract Prokaryotes evolved to thrive in an extremely diverse set of habitats, and their proteomes bear signatures of environmental conditions. Although correlations between amino acid usage and environmental temperature are well-documented, understanding of the mechanisms of thermal adaptation remains incomplete. Here, we couple the energetic costs of protein folding and protein homeostasis to build a microscopic model explaining both the overall amino acid composition and its temperature trends. Low biosynthesis costs lead to low diversity of physical interactions between amino acid residues, which in turn makes proteins less stable and drives up chaperone activity to maintain appropriate levels of folded, functional proteins. Assuming that the cost of chaperone activity is proportional to the fraction of unfolded client proteins, we simulated thermal adaptation of model proteins subject to minimization of the total cost of amino acid synthesis and chaperone activity. For the first time, we predicted both the proteome-average amino acid abundances and their temperature trends simultaneously, and found strong correlations between model predictions and 402 genomes of bacteria and archaea. The energetic constraint on protein evolution is more apparent in highly expressed proteins, selected by codon adaptation index. We found that in bacteria, highly expressed proteins are similar in composition to thermophilic ones, whereas in archaea no correlation between predicted expression level and thermostability was observed. At the same time, thermal adaptations of highly expressed proteins in bacteria and archaea are nearly identical, suggesting that universal energetic constraints prevail over the phylogenetic differences between these domains of life. PMID:29106597

[Construction of RNA-containing virus-like nanoparticles expression vector with cysteine residues on surface and fluorescent decoration].

Science.gov (United States)

Cheng, Yang-Jian; Liang, Ji-Xuan; Li, Qing-Ge

2005-08-01

Site-directed mutagenesis was performed at the codon 15 of the MS2 bacteriophage coat protein gene,which had been cloned to the virus-like particles expression vector containing non-self RNA fragment. The produced expression vector,termed pARSC, was transformed to E. coli DH5alpha. The positive clones were selected and proliferated. The harvested cells were treated with sonication and the supernatant was then subjected to linear sucrose density gradients centrifugation (15% to 60%) at 32000 r/min for 4 h at 4 degrees C. The virus-like particles, VLP-Cy, were collected at 35% sucrose density. The particles were examined by transmission electron microscopy and the spherical viral particles of approximately 27 nm in diameter were found. The thiolated VLP-Cy was then chemically modified with fluorescein -5'-maleimide. The covalent fluorescent labeling was confirmed by absorption analysis, SDS-PAGE and MALDI-TOF mass spectroscopy. This is the first report of preparation of RNA-containing natural fluorescent nanoparticles. The study highlight the versatility of MS2 bacteriophage capsids as building blocks for functional nanomaterials construction for a variety of application purposes.

Evaluation of a vectored equine herpesvirus type 1 (EHV-1) vaccine expressing H3 haemagglutinin in the protection of dogs against canine influenza.

Science.gov (United States)

Rosas, Cristina; Van de Walle, Gerlinde R; Metzger, Stephan M; Hoelzer, Karin; Dubovi, Edward J; Kim, Sung G; Parrish, Colin R; Osterrieder, Nikolaus

2008-05-02

In 2004, canine influenza virus (CIV) was identified as a respiratory pathogen of dogs for the first time and found to be closely related to H3N8 equine influenza virus (EIV). We generated a recombinant vectored vaccine that expresses H3 of a recent isolate of EIV using equine herpesvirus type 1 (EHV-1) as the delivery vehicle. This EHV-1 vectored vaccine exhibited robust and stable EIV H3 expression and induced a strong influenza virus-specific response in both mice and dogs upon intranasal or subcutaneous administration. Furthermore, upon challenge with the recent CIV isolate A/canine/PA/10915-07, protection of vaccinated dogs could be demonstrated by a significant reduction in clinical sings, and, more importantly, by a significant reduction in virus shedding. We concluded that the EHV-1/H3 recombinant vector can be a valuable alternative for protection of dogs against clinical disease induced by CIV and can significantly reduce virus spread.

Microbial biomass and viral infections of heterotrophic prokaryotes in the sub-surface layer of the central Arctic Ocean

Science.gov (United States)

Steward, Grieg F.; Fandino, Laura B.; Hollibaugh, James T.; Whitledge, Terry E.; Azam, Farooq

2007-10-01

Seawater samples were collected for microbial analyses between 55 and 235 m depth across the Arctic Ocean during the SCICEX 97 expedition (03 September-02 October 1997) using a nuclear submarine as a research platform. Abundances of prokaryotes (range 0.043-0.47×10 9 dm -3) and viruses (range 0.68-11×10 9 dm -3) were correlated ( r=0.66, n=150) with an average virus:prokaryote ratio of 26 (range 5-70). Biomass of prokaryotes integrated from 55 to 235 m ranged from 0.27 to 0.85 g C m -2 exceeding that of phytoplankton (0.005-0.2 g C m -2) or viruses (0.02-0.05 g C m -2) over the same depth range by an order of magnitude on average. Using transmission electron microscopy (TEM), we estimated that 0.5% of the prokaryote community on average (range 0-1.4%) was visibly infected with viruses, which suggests that very little of prokaryotic secondary production was lost due to viral lysis. Intracellular viruses ranged from 5 to >200/cell, with an average apparent burst size of 45±38 (mean±s.d.; n=45). TEM also revealed the presence of putative metal-precipitating bacteria in 8 of 13 samples, which averaged 0.3% of the total prokaryote community (range 0-1%). If these prokaryotes are accessible to protistan grazers, the Fe and Mn associated with their capsules might be an important source of trace metals to the planktonic food web. After combining our abundance and mortality data with data from the literature, we conclude that the biomass of prokaryoplankton exceeds that of phytoplankton when averaged over the upper 250 m of the central Arctic Ocean and that the fate of this biomass is poorly understood.

Generation of a vector system facilitating cloning of DMBT1 variants and recombinant expression of functional full-length DMBT1

DEFF Research Database (Denmark)

End, Caroline; Lyer, Stefan; Renner, Marcus

2005-01-01

of a vector system that facilitates cloning of DMBT1 variants. We demonstrate applicability of the vector system by expression of the largest DMBT1 variant in a tetracycline-inducible mammalian expression system using the Chinese hamster ovary cell line. Yields up to 30 mg rDMBT1 per litre of cell culture......Deleted in malignant brain tumours 1 (DMBT1) codes for a approximately 340kDa glycoprotein with highly repetitive scavenger receptor cysteine-rich (SRCR) domains. DMBT1 was implicated in cancer, defence against viral and bacterial infections, and differentiation of epithelial cells. Recombinant...... yields, and protein preparations which may substantially vary due to differential processing and genetic polymorphism, all of which impedes functional research on DMBT1. Cloning of DMBT1 cDNAs is hampered because of the size and the 13 highly homologous SRCR exons. In this study, we report on the setup...

Non-Random Inversion Landscapes in Prokaryotic Genomes Are Shaped by Heterogeneous Selection Pressures.

Science.gov (United States)

Repar, Jelena; Warnecke, Tobias

2017-08-01

Inversions are a major contributor to structural genome evolution in prokaryotes. Here, using a novel alignment-based method, we systematically compare 1,651 bacterial and 98 archaeal genomes to show that inversion landscapes are frequently biased toward (symmetric) inversions around the origin-terminus axis. However, symmetric inversion bias is not a universal feature of prokaryotic genome evolution but varies considerably across clades. At the extremes, inversion landscapes in Bacillus-Clostridium and Actinobacteria are dominated by symmetric inversions, while there is little or no systematic bias favoring symmetric rearrangements in archaea with a single origin of replication. Within clades, we find strong but clade-specific relationships between symmetric inversion bias and different features of adaptive genome architecture, including the distance of essential genes to the origin of replication and the preferential localization of genes on the leading strand. We suggest that heterogeneous selection pressures have converged to produce similar patterns of structural genome evolution across prokaryotes. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Constitutive and regulated expression vectors to construct polyphosphate deficient bacteria

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Jerez Carlos A

2009-03-01

Full Text Available Abstract Background Inorganic polyphosphate (polyP, a polymer of tens or hundreds of phosphate residues linked by ATP-like bonds, is found in all organisms and performs a wide variety of functions. PolyP is synthesized in bacterial cells by the actions of polyphosphate kinases (PPK1 and PPK2 and degraded by an exopolyphosphatase (PPX. Bacterial cells with polyP deficiencies are impaired in many structural and important cellular functions such as motility, quorum sensing, biofilm formation and virulence. Knockout mutants of the ppk1 gene have been the most frequent strategy employed to generate polyP deficient cells. Results As an alternative method to construct polyP-deficient bacteria we developed constitutive and regulated broad-host-range vectors for depleting the cellular polyP content. This was achieved by the overexpression of yeast exopolyphosphatase (PPX1. Using this approach in a polyphosphate accumulating bacteria (Pseudomonas sp. B4, we were able to eliminate most of the cellular polyP (>95%. Furthermore, the effect of overexpression of PPX1 resembled the functional defects found in motility and biofilm formation in a ppk1 mutant from Pseudomonas aeruginosa PAO1. The plasmids constructed were also successfully replicated in other bacteria such as Escherichia coli, Burkholderia and Salmonella. Conclusion To deplete polyP contents in bacteria broad-host-range expression vectors can be used as an alternative and more efficient method compared with the deletion of ppk genes. It is of great importance to understand why polyP deficiency affects vital cellular processes in bacteria. The construction reported in this work will be of great relevance to study the role of polyP in microorganisms with non-sequenced genomes or those in which orthologs to ppk genes have not been identified.

Translational up-regulation and high-level protein expression from plasmid vectors by mTOR activation via different pathways in PC3 and 293T cells.

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Prashanthi Karyala

Full Text Available BACKGROUND: Though 293T cells are widely used for expression of proteins from transfected plasmid vectors, the molecular basis for the high-level expression is yet to be understood. We recently identified the prostate carcinoma cell line PC3 to be as efficient as 293T in protein expression. This study was undertaken to decipher the molecular basis of high-level expression in these two cell lines. METHODOLOGY/PRINCIPAL FINDINGS: In a survey of different cell lines for efficient expression of platelet-derived growth factor-B (PDGF-B, β-galactosidase (β-gal and green fluorescent protein (GFP from plasmid vectors, PC3 was found to express at 5-50-fold higher levels compared to the bone metastatic prostate carcinoma cell line PC3BM and many other cell lines. Further, the efficiency of transfection and level of expression of the reporters in PC3 were comparable to that in 293T. Comparative analyses revealed that the high level expression of the reporters in the two cell lines was due to increased translational efficiency. While phosphatidic acid (PA-mediated activation of mTOR, as revealed by drastic reduction in reporter expression by n-butanol, primarily contributed to the high level expression in PC3, multiple pathways involving PA, PI3K/Akt and ERK1/2 appear to contribute to the abundant reporter expression in 293T. Thus the extent of translational up-regulation attained through the concerted activation of mTOR by multiple pathways in 293T could be achieved through its activation primarily by the PA pathway in PC3. CONCLUSIONS/SIGNIFICANCE: Our studies reveal that the high-level expression of proteins from plasmid vectors is effected by translational up-regulation through mTOR activation via different signaling pathways in the two cell lines and that PC3 is as efficient as 293T for recombinant protein expression. Further, PC3 offers an advantage in that the level of expression of the protein can be regulated by simple addition of n-butanol to

Augmentation of alphavirus vector-induced human papilloma virus-specific immune and anti-tumour responses by co-expression of interleukin-12

NARCIS (Netherlands)

Riezebos-Brilman, Annelies; Regts, Joke; Chen, Margaret; Wilschut, Jan; Daemen, Toos

2009-01-01

To enhance the efficacy of a therapeutic immunisition strategy against human papillomavirus-induced cervical cancer we evaluated the adjuvant effect of interleukin-12 (IL12) expressed by a Semliki Forest virus vector (SFV) in mice. Depending on the dose and schedule. SFV-IL12 Stimulated

A Lactococcus lactis expression vector set with multiple affinity tags to facilitate isolation and direct labeling of heterologous secreted proteins

NARCIS (Netherlands)

Pastrana, Francisco Romero; Neef, Jolanda; van Dijl, Jan Maarten; Buist, Girbe

The gram-positive bacterium Lactococcus lactis is a useful host for extracellular protein production. A main advantage of L. lactis over other bacterial expression systems is that lactococcal cells display low levels of autolysis and proteolysis. Previously, we developed a set of vectors for

Regulation of mtl operon promoter of Bacillus subtilis: requirements of its use in expression vectors

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Altenbuchner Josef

2011-10-01

Full Text Available Abstract Background Several vector systems have been developed to express any gene desired to be studied in Bacillus subtilis. Among them, the transcriptionally regulated promoters involved in carbohydrate utilization are a research priority. Expression systems based on Bacillus promoters for xylose, maltose, and mannose utilization, as well as on the heterologous E. coli lactose promoter, have been successfully constructed. The promoter of the mtlAFD operon for utilization of mannitol is another promising candidate for its use in expression vectors. In this study, we investigated the regulation of the mtl genes in order to identify the elements needed to construct a strong mannitol inducible expression system in B. subtilis. Results Regulation of the promoters of mtlAFD operon (PmtlA and mtlR (PmtlR encoding the activator were investigated by fusion to lacZ. Identification of the PmtlA and PmtlR transcription start sites revealed the σA like promoter structures. Also, the operator of PmtlA was determined by shortening, nucleotide exchange, and alignment of PmtlA and PmtlR operator regions. Deletion of the mannitol-specific PTS genes (mtlAF resulted in PmtlA constitutive expression demonstrating the inhibitory effect of EIICBMtl and EIIAMtl on MtlR in the absence of mannitol. Disruption of mtlD made the cells sensitive to mannitol and glucitol. Both PmtlA and PmtlR were influenced by carbon catabolite repression (CCR. However, a CcpA deficient mutant showed only a slight reduction in PmtlR catabolite repression. Similarly, using PgroE as a constitutive promoter, putative cre sites of PmtlA and PmtlR slightly reduced the promoter activity in the presence of glucose. In contrast, glucose repression of PmtlA and PmtlR was completely abolished in a ΔptsG mutant and significantly reduced in a MtlR (H342D mutant. Conclusions The mtl operon promoter (PmtlA is a strong promoter that reached a maximum of 13,000 Miller units with lacZ as a reporter on

Do marine natural products interfere with prokaryotic AHL regulatory systems?

DEFF Research Database (Denmark)

Kjelleberg, S.; Steinberg, P.; Givskov, Michael Christian

1997-01-01

Recent studies indicate that a taxonomically diverse range of marine eukaryotes produce metabolites which inhibit phenotypic traits in bacteria, with no or minimal effects on growth. In this review, we present evidence for the existence of such eukaryotic interference with a conserved prokaryotic...

RNA in defense: CRISPRs protect prokaryotes against mobile genetic elements

NARCIS (Netherlands)

Jore, M.M.; Brouns, S.J.J.; Oost, van der J.

2012-01-01

The CRISPR/Cas system in prokaryotes provides resistance against invading viruses and plasmids. Three distinct stages in the mechanism can be recognized. Initially, fragments of invader DNA are integrated as new spacers into the repetitive CRISPR locus. Subsequently, the CRISPR is transcribed and

Use of endophytic diazotrophic bacteria as a vector to express the cry3A gene from Bacillus thuringiensis

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Salles Joana Falcão

2000-01-01

Full Text Available The goal of this study was to evaluate the potential of endophytic diazotrophic bacteria as a vector to express a cry gene from Bacillus thuringiensis, envisaging the control of pests that attack sugarcane plants. The endophytic nitrogen-fixing bacteria Gluconacetobacter diazotrophicus strain BR11281 and Herbaspirillum seropedicae strain BR11335 were used as models. The cry3A gene was transferred by conjugation using a suicide plasmid and the recombinant strains were selected by their ability to fix nitrogen in semi-solid N-free medium. The presence of the cry gene was detected by Southern-blot using an internal fragment of 1.0 kb as a probe. The production of delta-endotoxin by the recombinant H. seropedicae strain was detected by dot blot while for G. diazotrophicus the Western-blot technique was used. In both cases, a specific antibody raised against the B. thuringiensis toxin was applied. The delta-endotoxin production showed by the G. diazotrophicus recombinant strain was dependent on the nitrogen fixing conditions since the cry3A gene was fused to a nif promoter. In the case of H. seropedicae the delta-endotoxin expression was not affected by the promoter (rhi used. These results suggest that endophytic diazotrophic bacteria can be used as vectors to express entomopathogenic genes envisaging control of sugarcane pests.

SIS: a program to generate draft genome sequence scaffolds for prokaryotes

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Dias Zanoni

2012-05-01

Full Text Available Abstract Background Decreasing costs of DNA sequencing have made prokaryotic draft genome sequences increasingly common. A contig scaffold is an ordering of contigs in the correct orientation. A scaffold can help genome comparisons and guide gap closure efforts. One popular technique for obtaining contig scaffolds is to map contigs onto a reference genome. However, rearrangements that may exist between the query and reference genomes may result in incorrect scaffolds, if these rearrangements are not taken into account. Large-scale inversions are common rearrangement events in prokaryotic genomes. Even in draft genomes it is possible to detect the presence of inversions given sufficient sequencing coverage and a sufficiently close reference genome. Results We present a linear-time algorithm that can generate a set of contig scaffolds for a draft genome sequence represented in contigs given a reference genome. The algorithm is aimed at prokaryotic genomes and relies on the presence of matching sequence patterns between the query and reference genomes that can be interpreted as the result of large-scale inversions; we call these patterns inversion signatures. Our algorithm is capable of correctly generating a scaffold if at least one member of every inversion signature pair is present in contigs and no inversion signatures have been overwritten in evolution. The algorithm is also capable of generating scaffolds in the presence of any kind of inversion, even though in this general case there is no guarantee that all scaffolds in the scaffold set will be correct. We compare the performance of sis, the program that implements the algorithm, to seven other scaffold-generating programs. The results of our tests show that sis has overall better performance. Conclusions sis is a new easy-to-use tool to generate contig scaffolds, available both as stand-alone and as a web server. The good performance of sis in our tests adds evidence that large

Design, Engineering, and Characterization of Prokaryotic Ligand-Binding Transcriptional Activators as Biosensors in Yeast

DEFF Research Database (Denmark)

Ambri, Francesca; Snoek, Tim; Skjødt, Mette Louise

2018-01-01

process. In the yeast Saccharomyces cerevisiae, implementation of allosterically regulated transcription factors from prokaryotes as metabolite biosensors has proven a valuable strategy to alleviate this screening bottleneck. Here, we present a protocol to select and incorporate prokaryotic...... transcriptional activators as metabolite biosensors in S. cerevisiae. As an example, we outline the engineering and characterization of the LysR-type transcriptional regulator (LTTR) family member BenM from Acetinobacter sp. ADP1 for monitoring accumulation of cis,cis-muconic acid, a bioplast precursor, in yeast...

Characterization of Recombinant Thermococcus kodakaraensis (KOD) DNA Polymerases Produced Using Silkworm-Baculovirus Expression Vector System

KAUST Repository

Yamashita, Mami

2017-05-08

The KOD DNA polymerase from Thermococcus kodakarensis (Tkod-Pol) has been preferred for PCR due to its rapid elongation rate, extreme thermostability and outstanding fidelity. Here in this study, we utilized silkworm-baculovirus expression vector system (silkworm-BEVS) to express the recombinant Tkod-Pol (rKOD) with N-terminal (rKOD-N) or C-terminal (rKOD-C) tandem fusion tags. By using BEVS, we produced functional rKODs with satisfactory yields, about 1.1 mg/larva for rKOD-N and 0.25 mg/larva for rKOD-C, respectively. Interestingly, we found that rKOD-C shows higher thermostability at 95 °C than that of rKOD-N, while that rKOD-N is significantly unstable after exposing to long period of heat-shock. We also assessed the polymerase activity as well as the fidelity of purified rKODs under various conditions. Compared with commercially available rKOD, which is expressed in E. coli expression system, rKOD-C exhibited almost the same PCR performance as the commercial rKOD did, while rKOD-N did lower performance. Taken together, our results suggested that silkworm-BEVS can be used to express and purify efficient rKOD in a commercial way.

Characterization of Recombinant Thermococcus kodakaraensis (KOD) DNA Polymerases Produced Using Silkworm-Baculovirus Expression Vector System

KAUST Repository

Yamashita, Mami; Xu, Jian; Morokuma, Daisuke; Hirata, Kazuma; Hino, Masato; Mon, Hiroaki; Takahashi, Masateru; Hamdan, Samir; Sakashita, Kosuke; Iiyama, Kazuhiro; Banno, Yutaka; Kusakabe, Takahiro; Lee, Jae Man

2017-01-01

The KOD DNA polymerase from Thermococcus kodakarensis (Tkod-Pol) has been preferred for PCR due to its rapid elongation rate, extreme thermostability and outstanding fidelity. Here in this study, we utilized silkworm-baculovirus expression vector system (silkworm-BEVS) to express the recombinant Tkod-Pol (rKOD) with N-terminal (rKOD-N) or C-terminal (rKOD-C) tandem fusion tags. By using BEVS, we produced functional rKODs with satisfactory yields, about 1.1 mg/larva for rKOD-N and 0.25 mg/larva for rKOD-C, respectively. Interestingly, we found that rKOD-C shows higher thermostability at 95 °C than that of rKOD-N, while that rKOD-N is significantly unstable after exposing to long period of heat-shock. We also assessed the polymerase activity as well as the fidelity of purified rKODs under various conditions. Compared with commercially available rKOD, which is expressed in E. coli expression system, rKOD-C exhibited almost the same PCR performance as the commercial rKOD did, while rKOD-N did lower performance. Taken together, our results suggested that silkworm-BEVS can be used to express and purify efficient rKOD in a commercial way.

[Description of the phylogenetic structure of hydrolytic prokaryotic complex in the soils].

Science.gov (United States)

Lukacheva, E G; Chernov, T I; Bykova, E M; Vlasenko, A N; Manucharova, N A

2013-01-01

With the help of the molecular-biological method of cell hybridization in situ (FISH), the abundance of a physiologically active hydrolytic prokaryotic complex in chernozem and gley-podzolic soils is determined. The total proportion of metabolically active cells, which were detected by hybridization with universal probes as representatives of the domains Bacteria and Archaea, in samples of the studied soil, was from 38% for chernozem up to 78% for gley-podzolic soil of the total number of cells. The differences in the structure of chitinolytic and pectinolytic prokaryotic soil complexes are detected. Along with the high abundance of Actinobacteria and Firmicutes in the soils with chitin, an increase in phylogenetic groups such as Alphaproteobacteria and Bacteroidetes is observed.

PSP: rapid identification of orthologous coding genes under positive selection across multiple closely related prokaryotic genomes.

Science.gov (United States)

Su, Fei; Ou, Hong-Yu; Tao, Fei; Tang, Hongzhi; Xu, Ping

2013-12-27

With genomic sequences of many closely related bacterial strains made available by deep sequencing, it is now possible to investigate trends in prokaryotic microevolution. Positive selection is a sub-process of microevolution, in which a particular mutation is favored, causing the allele frequency to continuously shift in one direction. Wide scanning of prokaryotic genomes has shown that positive selection at the molecular level is much more frequent than expected. Genes with significant positive selection may play key roles in bacterial adaption to different environmental pressures. However, selection pressure analyses are computationally intensive and awkward to configure. Here we describe an open access web server, which is designated as PSP (Positive Selection analysis for Prokaryotic genomes) for performing evolutionary analysis on orthologous coding genes, specially designed for rapid comparison of dozens of closely related prokaryotic genomes. Remarkably, PSP facilitates functional exploration at the multiple levels by assignments and enrichments of KO, GO or COG terms. To illustrate this user-friendly tool, we analyzed Escherichia coli and Bacillus cereus genomes and found that several genes, which play key roles in human infection and antibiotic resistance, show significant evidence of positive selection. PSP is freely available to all users without any login requirement at: http://db-mml.sjtu.edu.cn/PSP/. PSP ultimately allows researchers to do genome-scale analysis for evolutionary selection across multiple prokaryotic genomes rapidly and easily, and identify the genes undergoing positive selection, which may play key roles in the interactions of host-pathogen and/or environmental adaptation.

Cellular Viscosity in Prokaryotes and Thermal Stability of Low Molecular Weight Biomolecules.

Science.gov (United States)

Cuecas, Alba; Cruces, Jorge; Galisteo-López, Juan F; Peng, Xiaojun; Gonzalez, Juan M

2016-08-23

Some low molecular weight biomolecules, i.e., NAD(P)H, are unstable at high temperatures. The use of these biomolecules by thermophilic microorganisms has been scarcely analyzed. Herein, NADH stability has been studied at different temperatures and viscosities. NADH decay increased at increasing temperatures. At increasing viscosities, NADH decay rates decreased. Thus, maintaining relatively high cellular viscosity in cells could result in increased stability of low molecular weight biomolecules (i.e., NADH) at high temperatures, unlike what was previously deduced from studies in diluted water solutions. Cellular viscosity was determined using a fluorescent molecular rotor in various prokaryotes covering the range from 10 to 100°C. Some mesophiles showed the capability of changing cellular viscosity depending on growth temperature. Thermophiles and extreme thermophiles presented a relatively high cellular viscosity, suggesting this strategy as a reasonable mechanism to thrive under these high temperatures. Results substantiate the capability of thermophiles and extreme thermophiles (growth range 50-80°C) to stabilize and use generally considered unstable, universal low molecular weight biomolecules. In addition, this study represents a first report, to our knowledge, on cellular viscosity measurements in prokaryotes and it shows the dependency of prokaryotic cellular viscosity on species and growth temperature. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Once in a lifetime: strategies for preventing re-replication in prokaryotic and eukaryotic cells

DEFF Research Database (Denmark)

Nielsen, Olaf; Løbner-Olesen, Anders

2008-01-01

DNA replication is an extremely accurate process and cells have evolved intricate control mechanisms to ensure that each region of their genome is replicated only once during S phase. Here, we compare what is known about the processes that prevent re-replication in prokaryotic and eukaryotic cells...... prokaryotes and eukaryotes are inactivated until the next cell cycle. Furthermore, in both systems the beta-clamp of the replicative polymerase associates with enzymatic activities that contribute to the inactivation of the helicase loaders. Finally, recent studies suggest that the control mechanism...

An integrative and applicable phylogenetic footprinting framework for cis-regulatory motifs identification in prokaryotic genomes.

Science.gov (United States)

Liu, Bingqiang; Zhang, Hanyuan; Zhou, Chuan; Li, Guojun; Fennell, Anne; Wang, Guanghui; Kang, Yu; Liu, Qi; Ma, Qin

2016-08-09

Phylogenetic footprinting is an important computational technique for identifying cis-regulatory motifs in orthologous regulatory regions from multiple genomes, as motifs tend to evolve slower than their surrounding non-functional sequences. Its application, however, has several difficulties for optimizing the selection of orthologous data and reducing the false positives in motif prediction. Here we present an integrative phylogenetic footprinting framework for accurate motif predictions in prokaryotic genomes (MP(3)). The framework includes a new orthologous data preparation procedure, an additional promoter scoring and pruning method and an integration of six existing motif finding algorithms as basic motif search engines. Specifically, we collected orthologous genes from available prokaryotic genomes and built the orthologous regulatory regions based on sequence similarity of promoter regions. This procedure made full use of the large-scale genomic data and taxonomy information and filtered out the promoters with limited contribution to produce a high quality orthologous promoter set. The promoter scoring and pruning is implemented through motif voting by a set of complementary predicting tools that mine as many motif candidates as possible and simultaneously eliminate the effect of random noise. We have applied the framework to Escherichia coli k12 genome and evaluated the prediction performance through comparison with seven existing programs. This evaluation was systematically carried out at the nucleotide and binding site level, and the results showed that MP(3) consistently outperformed other popular motif finding tools. We have integrated MP(3) into our motif identification and analysis server DMINDA, allowing users to efficiently identify and analyze motifs in 2,072 completely sequenced prokaryotic genomes. The performance evaluation indicated that MP(3) is effective for predicting regulatory motifs in prokaryotic genomes. Its application may enhance

CpLEA5, the Late Embryogenesis Abundant Protein Gene from Chimonanthus praecox, Possesses Low Temperature and Osmotic Resistances in Prokaryote and Eukaryotes

Directory of Open Access Journals (Sweden)

Yiling Liu

2015-11-01

Full Text Available Plants synthesize and accumulate a series of stress-resistance proteins to protect normal physiological activities under adverse conditions. Chimonanthus praecox which blooms in freezing weather accumulates late embryogenesis abundant proteins (LEAs in flowers, but C. praecox LEAs are little reported. Here, we report a group of five LEA genes of C. praecox (CpLEA5, KT727031. Prokaryotic-expressed CpLEA5 was employed in Escherichia coli to investigate bioactivities and membrane permeability at low-temperature. In comparison with the vacant strains, CpLEA5-containing strains survived in a 20% higher rate; and the degree of cell membrane damage in CpLEA5-containing strains was 55% of that of the vacant strains according to a conductivity test, revealing the low-temperature resistance of CpLEA5 in bacteria. CpLEA5 was also expressed in Pichia pastoris. Interestingly, besides low-temperature resistance, CpLEA5 conferred high resistance to salt and alkali in CpLEA5 overexpressing yeast. The CpLEA5 gene was transferred into Arabidopsis thaliana to also demonstrate CpLEA5 actions in plants. As expected, the transgenic lines were more resistant against low-temperature and drought while compared with the wild type. Taken together, CpLEA5-conferred resistances to several conditions in prokaryote and eukaryotes could have great value as a genetic technology to enhance osmotic stress and low-temperature tolerance.

Bioinformatics analysis of disordered proteins in prokaryotes

Directory of Open Access Journals (Sweden)

Malkov Saša N

2011-03-01

Full Text Available Abstract Background A significant number of proteins have been shown to be intrinsically disordered, meaning that they lack a fixed 3 D structure or contain regions that do not posses a well defined 3 D structure. It has also been proven that a protein's disorder content is related to its function. We have performed an exhaustive analysis and comparison of the disorder content of proteins from prokaryotic organisms (i.e., superkingdoms Archaea and Bacteria with respect to functional categories they belong to, i.e., Clusters of Orthologous Groups of proteins (COGs and groups of COGs-Cellular processes (Cp, Information storage and processing (Isp, Metabolism (Me and Poorly characterized (Pc. We also analyzed the disorder content of proteins with respect to various genomic, metabolic and ecological characteristics of the organism they belong to. We used correlations and association rule mining in order to identify the most confident associations between specific modalities of the characteristics considered and disorder content. Results Bacteria are shown to have a somewhat higher level of protein disorder than archaea, except for proteins in the Me functional group. It is demonstrated that the Isp and Cp functional groups in particular (L-repair function and N-cell motility and secretion COGs of proteins in specific possess the highest disorder content, while Me proteins, in general, posses the lowest. Disorder fractions have been confirmed to have the lowest level for the so-called order-promoting amino acids and the highest level for the so-called disorder promoters. For each pair of organism characteristics, specific modalities are identified with the maximum disorder proteins in the corresponding organisms, e.g., high genome size-high GC content organisms, facultative anaerobic-low GC content organisms, aerobic-high genome size organisms, etc. Maximum disorder in archaea is observed for high GC content-low genome size organisms, high GC content

Circumventing antivector immunity: potential use of nonhuman adenoviral vectors.

Science.gov (United States)

Lopez-Gordo, Estrella; Podgorski, Iva I; Downes, Nicholas; Alemany, Ramon

2014-04-01

Adenoviruses are efficient gene delivery vectors based on their ability to transduce a wide variety of cell types and drive high-level transient transgene expression. While there have been advances in modifying human adenoviral (HAdV) vectors to increase their safety profile, there are still pitfalls that need to be further addressed. Preexisting humoral and cellular immunity against common HAdV serotypes limits the efficacy of gene transfer and duration of transgene expression. As an alternative, nonhuman AdV (NHAdV) vectors can circumvent neutralizing antibodies against HAdVs in immunized mice and monkeys and in human sera, suggesting that NHAdV vectors could circumvent preexisting humoral immunity against HAdVs in a clinical setting. Consequently, there has been an increased interest in developing NHAdV vectors for gene delivery in humans. In this review, we outline the recent advances and limitations of HAdV vectors for gene therapy and describe examples of NHAdV vectors focusing on their immunogenicity, tropism, and potential as effective gene therapy vehicles.

Relationship between viral and prokaryotic abundance on the Bajo O’Higgins 1 Seamount (Humboldt Current System off Chile

Directory of Open Access Journals (Sweden)

Oscar E. Chiang

2007-03-01

Full Text Available There is little known about the ecology of microbial communities living in the water column over seamounts. Here, for the first time, the spatial distribution and abundance of virus-like particles (VLP are described over a seamount. The association between VLP distribution, prokaryotic abundance, and environmental variables is also analyzed. Sampling was conducted in December 2004 on the Bajo O’Higgins 1 seamount (32°54’S, 73°53’W located in the Humboldt Current System off Chile. A oxygen minimum layer (OMZ was clearly present between 130 and 280 m in the water column over the seamount. Water samples were taken with Niskin bottles at 10 oceanographic stations over the seamount at depths of 5, 20, 50, 75, 100, and 150 m and at the benthic boundary layer (BBL; 5-12 m over the sediments. Temperature, salinity, oxygen, chlorophyll , and phaeopigments were measured at each station. Viral and prokaryotic abundances were determined with fluorochrome SYBR Green I. Viral abundance ranged from 1.53 x 109 VLP L-1 - 16.48 x 109 VLP L-1, whereas prokaryotic abundance ranged from 1.78 x 10 8 cell L-1 - 14.91 x 108 cell L-1. The virus-like particle/prokaryote ratio varied widely among the analyzed layers (i.e. surface, OMZ, and BBL, probably due to the presence of different prokaryotic and viral assemblages in each layer. Our results indicate that the environmental conditions, mainly the concentration of dissolved oxygen in the water column over Bajo O’Higgins 1 seamount, shape the association between viral and prokaryotic abundance.

Single vector system for efficient N-myristoylation of recombinant proteins in E. coli.

Directory of Open Access Journals (Sweden)

Julian M Glück

Full Text Available BACKGROUND: N-myristoylation is a crucial covalent modification of numerous eukaryotic and viral proteins that is catalyzed by N-myristoyltransferase (NMT. Prokaryotes are lacking endogenous NMT activity. Recombinant production of N-myristoylated proteins in E. coli cells can be achieved by coexpression of heterologous NMT with the target protein. In the past, dual plasmid systems were used for this purpose. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe a single vector system for efficient coexpression of substrate and enzyme suitable for production of co- or posttranslationally modified proteins. The approach was validated using the HIV-1 Nef protein as an example. A simple and efficient protocol for production of highly pure and completely N-myristoylated Nef is presented. The yield is about 20 mg myristoylated Nef per liter growth medium. CONCLUSIONS/SIGNIFICANCE: The single vector strategy allows diverse modifications of target proteins recombinantly coexpressed in E. coli with heterologous enzymes. The method is generally applicable and provides large amounts of quantitatively processed target protein that are sufficient for comprehensive biophysical and structural studies.

Biodiversity of prokaryotic communities associated with the ectoderm of Ectopleura crocea (Cnidaria, Hydrozoa.

Directory of Open Access Journals (Sweden)

Cristina Gioia Di Camillo

Full Text Available The surface of many marine organisms is colonized by complex communities of microbes, yet our understanding of the diversity and role of host-associated microbes is still limited. We investigated the association between Ectopleura crocea (a colonial hydroid distributed worldwide in temperate waters and prokaryotic assemblages colonizing the hydranth surface. We used, for the first time on a marine hydroid, a combination of electron and epifluorescence microscopy and 16S rDNA tag pyrosequencing to investigate the associated prokaryotic diversity. Dense assemblages of prokaryotes were associated with the hydrant surface. Two microbial morphotypes were observed: one horseshoe-shaped and one fusiform, worm-like. These prokaryotes were observed on the hydrozoan epidermis, but not in the portions covered by the perisarcal exoskeleton, and their abundance was higher in March while decreased in late spring. Molecular analyses showed that assemblages were dominated by Bacteria rather than Archaea. Bacterial assemblages were highly diversified, with up to 113 genera and 570 Operational Taxonomic Units (OTUs, many of which were rare and contributed to <0.4%. The two most abundant OTUs, likely corresponding to the two morphotypes present on the epidermis, were distantly related to Comamonadaceae (genus Delftia and to Flavobacteriaceae (genus Polaribacter. Epibiontic bacteria were found on E. crocea from different geographic areas but not in other hydroid species in the same areas, suggesting that the host-microbe association is species-specific. This is the first detailed report of bacteria living on the hydrozoan epidermis, and indeed the first study reporting bacteria associated with the epithelium of E. crocea. Our results provide a starting point for future studies aiming at clarifying the role of this peculiar hydrozoan-bacterial association.

Prokaryote genome fluidity: toward a system approach of the mobilome.

Science.gov (United States)

Toussaint, Ariane; Chandler, Mick

2012-01-01

The importance of horizontal/lateral gene transfer (LGT) in shaping the genomes of prokaryotic organisms has been recognized in recent years as a result of analysis of the increasing number of available genome sequences. LGT is largely due to the transfer and recombination activities of mobile genetic elements (MGEs). Bacterial and archaeal genomes are mosaics of vertically and horizontally transmitted DNA segments. This generates reticulate relationships between members of the prokaryotic world that are better represented by networks than by "classical" phylogenetic trees. In this review we summarize the nature and activities of MGEs, and the problems that presently limit their analysis on a large scale. We propose routes to improve their annotation in the flow of genomic and metagenomic sequences that currently exist and those that become available. We describe network analysis of evolutionary relationships among some MGE categories and sketch out possible developments of this type of approach to get more insight into the role of the mobilome in bacterial adaptation and evolution.

A novel technology to target adenovirus vectors : application in cells involved in atherosclerosis

NARCIS (Netherlands)

Gras, Jan Cornelis Emile

2007-01-01

In this thesis a novel technology is described to target adenovirus vectors. Adenovirus vectors are powerful tools to modulate gene expression. The use of these vectors however, is hampered by the fact that many for gene therapy interesting cell types do not, or only at low levels express the CAR

Construction of expression vector containing glnA gene and detection of NPT II activity in the transgenic rice calli using 32P-labelled compound

International Nuclear Information System (INIS)

Su Jin; Zhang Xueqin; Yan Qiusheng; Chen Zhangliang; You Chongbiao

1993-08-01

The glnA gene encoding glutamine synthetase (GS) was amplified from Azospirillum brasilense Sp7 by PCR technique. the amplified 1.4 kb DNA fragment was cloned at the EcoRV site of Bluescript-SK. Both sequencing and restriction digestion data showed that the 1.4 kb DNA fragment flanked with BamHI site at each end was really the glnA gene of A. brasilense Sp7. The glnA gene was ligated with Bg1 II site of pCo24. As a result, an expression vector pGSC35 with CaMV35S promoter was obtained. Using colony in situ hybridization with α- 32 P-dATP labelled probes to screen the positive clones, another glnA gene expression vector pAGNB92 with rice actin 1 promoter was constructed after three rounds of ligation and transformation. Protoplasts isolated from rice cell suspension line cv. T986 were transformed with glnA expression vectors pGSC35 and pAGNB92 containing neomycin phosphotransferase II (NPTII) gene by using PEG fusion and electroporation. Transformed microcalli were selected on media containing G418 disulfate salt. NPT II activity was detected in 37% of G418 resistant calli by using dot blot hybridization with γ- 32 P-ATP and kanamycin as substrate

Support vector machine classification and validation of cancer tissue samples using microarray expression data.

Science.gov (United States)

Furey, T S; Cristianini, N; Duffy, N; Bednarski, D W; Schummer, M; Haussler, D

2000-10-01

DNA microarray experiments generating thousands of gene expression measurements, are being used to gather information from tissue and cell samples regarding gene expression differences that will be useful in diagnosing disease. We have developed a new method to analyse this kind of data using support vector machines (SVMs). This analysis consists of both classification of the tissue samples, and an exploration of the data for mis-labeled or questionable tissue results. We demonstrate the method in detail on samples consisting of ovarian cancer tissues, normal ovarian tissues, and other normal tissues. The dataset consists of expression experiment results for 97,802 cDNAs for each tissue. As a result of computational analysis, a tissue sample is discovered and confirmed to be wrongly labeled. Upon correction of this mistake and the removal of an outlier, perfect classification of tissues is achieved, but not with high confidence. We identify and analyse a subset of genes from the ovarian dataset whose expression is highly differentiated between the types of tissues. To show robustness of the SVM method, two previously published datasets from other types of tissues or cells are analysed. The results are comparable to those previously obtained. We show that other machine learning methods also perform comparably to the SVM on many of those datasets. The SVM software is available at http://www.cs. columbia.edu/ approximately bgrundy/svm.

Prokaryotic responses to ammonium and organic carbon reveal alternative CO2 fixation pathways and importance of alkaline phosphatase in the mesopelagic North Atlantic

Directory of Open Access Journals (Sweden)

Federico Baltar

2016-10-01

Full Text Available To decipher the response of mesopelagic prokaryotic communities to input of nutrients, we tracked changes in prokaryotic abundance, extracellular enzymatic activities, heterotrophic production, dark dissolved inorganic carbon (DIC fixation, community composition (16S rRNA sequencing and community gene expression (metatranscriptomics in 3 microcosm experiments with water from the mesopelagic North Atlantic. Responses in 3 different treatments amended with thiosulfate, ammonium or organic matter (i.e. pyruvate plus acetate were compared to unamended controls. The strongest stimulation was found in the organic matter enrichments, where all measured rates increased >10-fold. Strikingly, in the organic matter treatment, the dark DIC fixation rates —assumed to be related to autotrophic metabolisms— were equally stimulated as all the other heterotrophic-related parameters. This increase in DIC fixation rates was paralleled by an up-regulation of genes involved in DIC assimilation via anaplerotic pathways. Alkaline phosphatase was the metabolic rate most strongly stimulated and its activity seemed to be related to cross-activation by nonpartner histidine kinases, and/or the activation of genes involved in the regulation of elemental balance during catabolic processes. These findings suggest that episodic events such as strong sedimentation of organic matter into the mesopelagic might trigger rapid increases of originally rare members of the prokaryotic community, enhancing heterotrophic and autotrophic carbon uptake rates, ultimately affecting carbon cycling. Our experiments highlight a number of fairly unstudied microbial processes of potential importance in mesopelagic waters that require future attention.

Protein expression vector and secretion signal peptide optimization to drive the production, secretion, and functional expression of the bacteriocin enterocin A in lactic acid bacteria.

Science.gov (United States)

Borrero, Juan; Jiménez, Juan J; Gútiez, Loreto; Herranz, Carmen; Cintas, Luis M; Hernández, Pablo E

2011-10-20

Replacement of the leader sequence (LS) of the bacteriocin enterocin A (LS(entA)) by the signal peptides (SP) of the protein Usp45 (SP(usp45)), and the bacteriocins enterocin P (SP(entP)), and hiracin JM79 (SP(hirJM79)) permits the production, secretion, and functional expression of EntA by different lactic acid bacteria (LAB). Chimeric genes encoding the SP(usp45), the SP(entP), and the SP(hirJM79) fused to mature EntA plus the EntA immunity genes (entA+entiA) were cloned into the expression vectors pNZ8048 and pMSP3545, under control of the inducible P(nisA) promoter, and in pMG36c, under control of the constitutive P(32) promoter. The amount, antimicrobial activity, and specific antimicrobial activity of the EntA produced by the recombinant Lactococcus lactis, Enterococcus faecium, E. faecalis, Lactobacillus sakei and Pediococcus acidilactici hosts varied depending on the signal peptide, the expression vector, and the host strain. However, the antimicrobial activity and the specific antimicrobial activity of the EntA produced by most of the LAB transformants was lower than expected from their production. The supernatants of the recombinant L. lactis NZ9000 (pNZUAI) and L. lactis NZ9000 (pNZHAI), overproducers of EntA, showed a 1.2- to 5.1-fold higher antimicrobial activity than that of the natural producer E. faecium T136 against different Listeria spp. Copyright © 2011 Elsevier B.V. All rights reserved.

Detecting uber-operons in prokaryotic genomes.

Science.gov (United States)

Che, Dongsheng; Li, Guojun; Mao, Fenglou; Wu, Hongwei; Xu, Ying

2006-01-01

We present a study on computational identification of uber-operons in a prokaryotic genome, each of which represents a group of operons that are evolutionarily or functionally associated through operons in other (reference) genomes. Uber-operons represent a rich set of footprints of operon evolution, whose full utilization could lead to new and more powerful tools for elucidation of biological pathways and networks than what operons have provided, and a better understanding of prokaryotic genome structures and evolution. Our prediction algorithm predicts uber-operons through identifying groups of functionally or transcriptionally related operons, whose gene sets are conserved across the target and multiple reference genomes. Using this algorithm, we have predicted uber-operons for each of a group of 91 genomes, using the other 90 genomes as references. In particular, we predicted 158 uber-operons in Escherichia coli K12 covering 1830 genes, and found that many of the uber-operons correspond to parts of known regulons or biological pathways or are involved in highly related biological processes based on their Gene Ontology (GO) assignments. For some of the predicted uber-operons that are not parts of known regulons or pathways, our analyses indicate that their genes are highly likely to work together in the same biological processes, suggesting the possibility of new regulons and pathways. We believe that our uber-operon prediction provides a highly useful capability and a rich information source for elucidation of complex biological processes, such as pathways in microbes. All the prediction results are available at our Uber-Operon Database: http://csbl.bmb.uga.edu/uber, the first of its kind.

Endothelial Cell-Targeted Adenoviral Vector for Suppressing Breast Malignancies

National Research Council Canada - National Science Library

Huang, Shuang

2004-01-01

.... Our proposal is designed to develop an endothelial cell-targeted adenoviral vector and to use the targeted vector to express high levels of anticancer therapeutic genes in the sites of angiogenenic...

Design, Construction, and Validation of Artificial MicroRNA Vectors Using Agrobacterium-Mediated Transient Expression System.

Science.gov (United States)

Bhagwat, Basdeo; Chi, Ming; Han, Dianwei; Tang, Haifeng; Tang, Guiliang; Xiang, Yu

2016-01-01

Artificial microRNA (amiRNA) technology utilizes microRNA (miRNA) biogenesis pathway to produce artificially selected small RNAs using miRNA gene backbone. It provides a feasible strategy for inducing loss of gene function, and has been applied in functional genomics study, improvement of crop quality and plant virus disease resistance. A big challenge in amiRNA applications is the unpredictability of silencing efficacy of the designed amiRNAs and not all constructed amiRNA candidates would be expressed effectively in plant cells. We and others found that high efficiency and specificity in RNA silencing can be achieved by designing amiRNAs with perfect or almost perfect sequence complementarity to their targets. In addition, we recently demonstrated that Agrobacterium-mediated transient expression system can be used to validate amiRNA constructs, which provides a simple, rapid and effective method to select highly expressible amiRNA candidates for stable genetic transformation. Here, we describe the methods for design of amiRNA candidates with perfect or almost perfect base-pairing to the target gene or gene groups, incorporation of amiRNA candidates in miR168a gene backbone by one step inverse PCR amplification, construction of plant amiRNA expression vectors, and assay of transient expression of amiRNAs in Nicotiana benthamiana through agro-infiltration, small RNA extraction, and amiRNA Northern blot.

[Construction and functional identification of eukaryotic expression vector carrying Sprague-Dawley rat MSX-2 gene].

Science.gov (United States)

Yang, Xian-Xian; Zhang, Mei; Yan, Zhao-Wen; Zhang, Ru-Hong; Mu, Xiong-Zheng

2008-01-01

To construct a high effective eukaryotic expressing plasmid PcDNA 3.1-MSX-2 encoding Sprague-Dawley rat MSX-2 gene for the further study of MSX-2 gene function. The full length SD rat MSX-2 gene was amplified by PCR, and the full length DNA was inserted in the PMD1 8-T vector. It was isolated by restriction enzyme digest with BamHI and Xhol, then ligated into the cloning site of the PcDNA3.1 expression plasmid. The positive recombinant was identified by PCR analysis, restriction endonudease analysis and sequence analysis. Expression of RNA and protein was detected by RT-PCR and Western blot analysis in PcDNA3.1-MSX-2 transfected HEK293 cells. Sequence analysis and restriction endonudease analysis of PcDNA3.1-MSX-2 demonstrated that the position and size of MSX-2 cDNA insertion were consistent with the design. RT-PCR and Western blot analysis showed specific expression of mRNA and protein of MSX-2 in the transfected HEK293 cells. The high effective eukaryotic expression plasmid PcDNA3.1-MSX-2 encoding Sprague-Dawley Rat MSX-2 gene which is related to craniofacial development can be successfully reconstructed. It may serve as the basis for the further study of MSX-2 gene function.

Use of prokaryotic transcriptional activators as metabolite biosensors in eukaryotic cells

DEFF Research Database (Denmark)

2018-01-01

The present invention relates to the use of transcriptional activators from prokaryotic organisms for use in eukaryotic cells, such as yeast as sensors of intracellular and extracellular accumulation of a ligand or metabolite specifically activating this transcriptional activator in a eukaryot...

Emerging experimental and computational technologies for purpose designed engineering of photosynthetic prokaryotes

KAUST Repository

Lindblad, Peter

2016-01-25

With recent advances in synthetic molecular tools to be used in photosynthetic prokaryotes, like cyanobacteria, it is possible to custom design and construct microbial cells for specific metabolic functions. This cross-disciplinary area of research has emerged within the interfaces of advanced genetic engineering, computational science, and molecular biotechnology. We have initiated the development of a genetic toolbox, using a synthetic biology approach, to custom design, engineer and construct cyanobacteria for selected function and metabolism. One major bottleneck is a controlled transcription and translation of introduced genetic constructs. An additional major issue is genetic stability. I will present and discuss recent progress in our development of genetic tools for advanced cyanobacterial biotechnology. Progress on understanding the electron pathways in native and engineered cyanobacterial enzymes and heterologous expression of non-native enymzes in cyanobacterial cells will be highlighted. Finally, I will discuss our attempts to merge synthetic biology with synthetic chemistry to explore fundamantal questions of protein design and function.

Construction of Expression Vector for Anti-Alpha-Fetoprotein Gene and Its Inhibition Effects on Alpha-Fetoprotein Positive Hepg2 Cells

Science.gov (United States)

Wang, Ze; Zhang, Hui

As research previously demonstrated, suppression of AFP expression or its biological activities might inhibit the proliferation of AFP positive human hepatocellular carcinoma cells. In this study, we constructed an anti-AFP gene vector and transfected it to HepG2 cells. RT-PCR showed AFP gene expression in the transfected cells was reduced. MTT assay suggested the proliferation of the transfected cells was also inhibited comparing with the untransfected cells. This result provides a new insight into AFP as the target for preventing and treating hepatocellular carcinoma.

Molecular cloning, expression, and in silico structural analysis of guinea pig IL-17.

Science.gov (United States)

Dirisala, Vijaya R; Jeevan, Amminikutty; Ramasamy, Suresh K; McMurray, David N

2013-11-01

Interleukin-17A (IL-17A) is a potent proinflammatory cytokine and the signature cytokine of Th17 cells, a subset which is involved in cytokine and chemokine production, neutrophil recruitment, promotion of T cell priming, and antibody production. IL-17 may play an important role in tuberculosis and other infectious diseases. In preparation for investigating its role in the highly relevant guinea pig model of pulmonary tuberculosis, we cloned guinea pig IL-17A for the first time. The complete coding sequence of the guinea pig IL-17A gene (477 nucleotides; 159 amino acids) was subcloned into a prokaryotic expression vector (pET-30a) resulting in the expression of a 17 kDa recombinant guinea pig IL-17A protein which was confirmed by mass spectrometry analysis. Homology modeling of guinea pig IL-17A revealed that the three-dimensional structure resembles that of human IL-17A. The secondary structure predicted for this protein showed the presence of one extra helix in the N-terminal region. The expression profile of IL-17A was analyzed quantitatively in spleen, lymph node, and lung cells from BCG-vaccinated guinea pigs by real-time PCR. The guinea pig IL-17A cDNA and its recombinant protein will serve as valuable tools for molecular and immunological studies in the guinea pig model of pulmonary TB and other human diseases.

Lateral gene transfer between prokaryotes and multicellular eukaryotes: ongoing and significant?

NARCIS (Netherlands)

Ros, V.I.D.; Hurst, G.D.D.

2009-01-01

The expansion of genome sequencing projects has produced accumulating evidence for lateral transfer of genes between prokaryotic and eukaryotic genomes. However, it remains controversial whether these genes are of functional importance in their recipient host. Nikoh and Nakabachi, in a recent paper

Novel thiols of prokaryotes.

Science.gov (United States)

Fahey, R C

2001-01-01

Glutathione metabolism is associated with oxygenic cyanobacteria and the oxygen-utilizing purple bacteria, but is absent in many other prokaryotes. This review focuses on novel thiols found in those bacteria lacking glutathione. Included are glutathione amide and its perthiol, produced by phototrophic purple sulfur bacteria and apparently involved in their sulfide metabolism. Among archaebacteria, coenzyme M (2-mercaptoethanesulfonic acid) and coenzyme B (7-mercaptoheptanoylthreonine phosphate) play central roles in the anaerobic production of CH4 and associated energy conversion by methanogens, whereas the major thiol in the aerobic phototrophic halobacteria is gamma-glutamylcysteine. The highly aerobic actinomycetes produce mycothiol, a conjugate of N-acetylcysteine with a pseudodisaccharide of glucosamine and myo-inositol, AcCys-GlcNalpha(1 --> 1)Ins, which appears to play an antioxidant role similar to glutathione. Ergothioneine, also produced by actinomycetes, remains a mystery despite many years of study. Available data on the biosynthesis and metabolism of these and other novel thiols is summarized and key areas for additional study are identified.

SURVIVAL AND EVOLUTION OF CRISPR-CAS SYSTEM IN PROKARYOTES AND ITS APPLICATIONS

Directory of Open Access Journals (Sweden)

Muhammad Abu Bakr Shabbir

2016-09-01

Full Text Available Prokaryotes have developed numerous innate immune mechanisms in order to fend off bacteriophage or plasmid attack. One of these immune systems is Clustered regularly interspaced short palindromic repeats (CRISPR. CRISPR associated proteins play a key role in survival of prokaryotes against invaders, as these systems cleave DNA of foreign genetic elements. Beyond providing immunity, these systems have significant impact in altering the bacterial physiology in term of its virulence and pathogenicity, as well as evolution. Also, due to their diverse nature of functionality, cas9 endoribonuclease can be easily reprogrammed with the help of guide RNAs, showing unprecedented potential and significance for gene editing in treating genetic diseases. Here, we also discuss the use of NgAgo-gDNA system in genome editing of human cells.

Survival and Evolution of CRISPR–Cas System in Prokaryotes and Its Applications

Science.gov (United States)

Shabbir, Muhammad Abu Bakr; Hao, Haihong; Shabbir, Muhammad Zubair; Hussain, Hafiz Iftikhar; Iqbal, Zahid; Ahmed, Saeed; Sattar, Adeel; Iqbal, Mujahid; Li, Jun; Yuan, Zonghui

2016-01-01

Prokaryotes have developed numerous innate immune mechanisms in order to fend off bacteriophage or plasmid attack. One of these immune systems is clustered regularly interspaced short palindromic repeats (CRISPR). CRISPR-associated proteins play a key role in survival of prokaryotes against invaders, as these systems cleave DNA of foreign genetic elements. Beyond providing immunity, these systems have significant impact in altering the bacterial physiology in term of its virulence and pathogenicity, as well as evolution. Also, due to their diverse nature of functionality, cas9 endoribonuclease can be easily reprogrammed with the help of guide RNAs, showing unprecedented potential and significance for gene editing in treating genetic diseases. Here, we also discuss the use of NgAgo–gDNA system in genome editing of human cells. PMID:27725818

The current status of cyanobacterial nomenclature under the "prokaryotic" and the "botanical" code.

Science.gov (United States)

Oren, Aharon; Ventura, Stefano

2017-10-01

Cyanobacterial taxonomy developed in the botanical world because Cyanobacteria/Cyanophyta have traditionally been identified as algae. However, they possess a prokaryotic cell structure, and phylogenetically they belong to the Bacteria. This caused nomenclature problems as the provisions of the International Code of Nomenclature for algae, fungi, and plants (ICN; the "Botanical Code") differ from those of the International Code of Nomenclature of Prokaryotes (ICNP; the "Prokaryotic Code"). While the ICN recognises names validly published under the ICNP, Article 45(1) of the ICN has not yet been reciprocated in the ICNP. Different solutions have been proposed to solve the current problems. In 2012 a Special Committee on the harmonisation of the nomenclature of Cyanobacteria was appointed, but its activity has been minimal. Two opposing proposals to regulate cyanobacterial nomenclature were recently submitted, one calling for deletion of the cyanobacteria from the groups of organisms whose nomenclature is regulated by the ICNP, the second to consistently apply the rules of the ICNP to all cyanobacteria. Following a general overview of the current status of cyanobacterial nomenclature under the two codes we present five case studies of genera for which nomenclatural aspects have been discussed in recent years: Microcystis, Planktothrix, Halothece, Gloeobacter and Nostoc.

An introduction to vectors, vector operators and vector analysis

CERN Document Server

Joag, Pramod S

2016-01-01

Ideal for undergraduate and graduate students of science and engineering, this book covers fundamental concepts of vectors and their applications in a single volume. The first unit deals with basic formulation, both conceptual and theoretical. It discusses applications of algebraic operations, Levi-Civita notation, and curvilinear coordinate systems like spherical polar and parabolic systems and structures, and analytical geometry of curves and surfaces. The second unit delves into the algebra of operators and their types and also explains the equivalence between the algebra of vector operators and the algebra of matrices. Formulation of eigen vectors and eigen values of a linear vector operator are elaborated using vector algebra. The third unit deals with vector analysis, discussing vector valued functions of a scalar variable and functions of vector argument (both scalar valued and vector valued), thus covering both the scalar vector fields and vector integration.

Analyzing the Differences and Preferences of Pathogenic and Nonpathogenic Prokaryote Species

Science.gov (United States)

Nolen, L.; Duong, K.; Heim, N. A.; Payne, J.

2015-12-01

A limited amount of knowledge exists on the large-scale characteristics and differences of pathogenic species in comparison to all prokaryotes. Pathogenic species, like other prokaryotes, have attributes specific to their environment and lifestyles. However, because they have evolved to coexist inside their hosts, the conditions they occupy may be more limited than those of non-pathogenic species. In this study we investigate the possibility of divergent evolution between pathogenic and non-pathogenic species by examining differences that may have evolved as a result of the need to adapt to their host. For this research we analyzed data collected from over 1900 prokaryotic species and performed t-tests using R to quantify potential differences in preferences. To examine the possible divergences from nonpathogenic bacteria, we focused on three variables: cell biovolume, preferred environmental pH, and preferred environmental temperature. We also looked at differences between pathogenic and nonpathogenic species belonging to the same phylum. Our results suggest a strong divergence in abiotic preferences between the two groups, with pathogens occupying a much smaller range of temperatures and pHs than their non-pathogenic counterparts. However, while the median biovolume is different when comparing pathogens and nonpathogens, we cannot conclude that the mean values are significantly different from each other. In addition, we found evidence of convergent evolution, as the temperature and pH preferences of pathogenic bacteria species from different phlya all approach the same values. Pathogenic species do not, however, all approach the same biovolume values, suggesting that specific pH and temperature preferences are more characteristic of pathogens than certain biovolumes.

[Construction and selection of effective mouse Smad6 recombinant lenti-virus interference vectors].

Science.gov (United States)

Yu, Jing; Qi, Mengchun; Deng, Jiupeng; Liu, Gang; Chen, Huaiqing

2010-10-01

This experiment was designed to construct mouse Smad6 recombinant RNA interference vectors and determine their interference effects on bone marrow mesenchymal stem cells (BMSCs). Three recombinant Smad6 RNA interference vectors were constructed by molecular clone techniques with a lenti-virus vector expressing green fluorescent protein (GFP), and the correctness of recombinant vectors was verified by DNA sequencing. Mouse BMSCs were used for transfection experiments and BMP-2 was in use for osteogenic induction of MSCs. The transfection efficiency of recombinant vectors was examined by Laser confocal scanning microscope and the interference effect of recombinant vectors on Smad6 gene expression was determined by real-time RT-PCR and Western blot, respectively. Three Smad6 recombinant RNA interference vectors were successfully constructed and their correctness was proved by DNA sequencing. After transfection, GFPs were effectively expressed in MSCs and all of three recombinant vectors gained high transfection efficiency (> 95%). Both real-time PCR and Western blot examination indicated that among three recombinant vectors, No. 2 Svector had the best interference effect and the interference effect was nearly 91% at protein level. In conclusion, Mouse recombinant Smad6 RNA interference (RNAi) vector was successfully constructed and it provided an effective tool for further studies on BMP signal pathways.

Emerging experimental and computational technologies for purpose designed engineering of photosynthetic prokaryotes

KAUST Repository

Lindblad, Peter

2016-01-01

With recent advances in synthetic molecular tools to be used in photosynthetic prokaryotes, like cyanobacteria, it is possible to custom design and construct microbial cells for specific metabolic functions. This cross-disciplinary area of research

A comparative analysis of constitutive promoters located in adeno-associated viral vectors.

Directory of Open Access Journals (Sweden)

Lkhagvasuren Damdindorj

Full Text Available The properties of constitutive promoters within adeno-associated viral (AAV vectors have not yet been fully characterized. In this study, AAV vectors, in which enhanced GFP expression was directed by one of the six constitutive promoters (human β-actin, human elongation factor-1α, chicken β-actin combined with cytomegalovirus early enhancer, cytomegalovirus (CMV, simian virus 40, and herpes simplex virus thymidine kinase, were constructed and introduced into the HCT116, DLD-1, HT-1080, and MCF-10A cell lines. Quantification of GFP signals in infected cells demonstrated that the CMV promoter produced the highest GFP expression in the six promoters and maintained relatively high GFP expression for up to eight weeks after infection of HCT116, DLD-1, and HT-1080. Exogenous human CDKN2A gene expression was also introduced into DLD-1 and MCF-10A in a similar pattern by using AAV vectors bearing the human β-actin and the CMV promoters. The six constitutive promoters were subsequently placed upstream of the neomycin resistance gene within AAV vectors, and HCT116, DLD-1, and HT-1080 were infected with the resulting vectors. Of the six promoters, the CMV promoter produced the largest number of G418-resistant colonies in all three cell lines. Because AAV vectors have been frequently used as a platform to construct targeting vectors that permit gene editing in human cell lines, we lastly infected the three cell lines with AAV-based targeting vectors against the human PIGA gene in which one of the six promoters regulate the neomycin resistance gene. This assay revealed that the CMV promoter led to the lowest PIGA gene targeting efficiency in the investigated promoters. These results provide a clue to the identification of constitutive promoters suitable to express exogenous genes with AAV vectors, as well as those helpful to conduct efficient gene targeting using AAV-based targeting vectors in human cell lines.

Energy Coupling Factor-Type ABC Transporters for Vitamin Uptake in Prokaryotes

NARCIS (Netherlands)

Erkens, Guus B.; Dosz-Majsnerowska, Maria; ter Beek, Josy; Slotboom, Dirk Jan

2012-01-01

Energy coupling factor (ECF) transporters are a subgroup of ATP-binding cassette (ABC) transporters involved in the uptake of vitamins and micronutrients in prokaryotes. In contrast to classical ABC importers, ECF transporters do not make use of water-soluble substrate binding proteins or domains

Focus on Membrane Differentiation and Membrane Domains in the Prokaryotic Cell

NARCIS (Netherlands)

Boekema, Egbert J.; Scheffers, Dirk-Jan; van Bezouwen, Laura S.; Bolhuis, Henk; Folea, I. Mihaela

2013-01-01

A summary is presented of membrane differentiation in the prokaryotic cell, with an emphasis on the organization of proteins in the plasma/cell membrane. Many species belonging to the Eubacteria and Archaea have special membrane domains and/or membrane proliferation, which are vital for different

Construction and Antiapoptosis Activities of Recombinant Adenoviral Expression Vector Carrying EBV Latent Membrane Protein 2A

Directory of Open Access Journals (Sweden)

Xishuang Liu

2011-01-01

Full Text Available To evaluate the possible effects of LMP2A (EBV latent membrane protein 2A on human gastric cancer cell line SGC-7901, LMP2A coding gene was subcloned into shuttle plasmid pAdTrackCMV to form transfer plasmid pAdTrackCMV-2A, which was linearized with PmeI and cotransformed into E.coli BJ5183 with adenovirus genomic plasmid of pAdeasy-1. The identified recombinant adenovirus plasmid DNA was digested with PacI and transfected into 293 cells to package recombinant adenovirus particles named vAd-2A. Then the expression and antiapoptosis activities of LMP2A on SGC-7901 infected with vAd-2A were analyzed. The vAd-2A was successfully constructed and identified by PCR, restriction digestion, and sequencing. LMP2A expression in SGC was identified by strong green fluorescence expression with fluorescence microscopic photograph and Southern blotting. The growth of LMP2A expressing SGC cells was apparently improved. Both cyclin E expression and S phase ratio in LMP2A expressing SGC cells were upregulated by cell cycle analysis and confocal microscopic analysis respectively. The replication-deficient recombinant adenovirus vector can express LMP2A antigen in SGC cells and inhibit their apoptosis. The results indicate that LMP2A might play an important role in pathogenesis of EBV-associated gastric cancer (EBVaGC. This study establishes a foundation for further study on EBVaGC and its gene therapy.

Genomes in turmoil: quantification of genome dynamics in prokaryote supergenomes.

Science.gov (United States)

Puigbò, Pere; Lobkovsky, Alexander E; Kristensen, David M; Wolf, Yuri I; Koonin, Eugene V

2014-08-21

Genomes of bacteria and archaea (collectively, prokaryotes) appear to exist in incessant flux, expanding via horizontal gene transfer and gene duplication, and contracting via gene loss. However, the actual rates of genome dynamics and relative contributions of different types of event across the diversity of prokaryotes are largely unknown, as are the sizes of microbial supergenomes, i.e. pools of genes that are accessible to the given microbial species. We performed a comprehensive analysis of the genome dynamics in 35 groups (34 bacterial and one archaeal) of closely related microbial genomes using a phylogenetic birth-and-death maximum likelihood model to quantify the rates of gene family gain and loss, as well as expansion and reduction. The results show that loss of gene families dominates the evolution of prokaryotes, occurring at approximately three times the rate of gain. The rates of gene family expansion and reduction are typically seven and twenty times less than the gain and loss rates, respectively. Thus, the prevailing mode of evolution in bacteria and archaea is genome contraction, which is partially compensated by the gain of new gene families via horizontal gene transfer. However, the rates of gene family gain, loss, expansion and reduction vary within wide ranges, with the most stable genomes showing rates about 25 times lower than the most dynamic genomes. For many groups, the supergenome estimated from the fraction of repetitive gene family gains includes about tenfold more gene families than the typical genome in the group although some groups appear to have vast, 'open' supergenomes. Reconstruction of evolution for groups of closely related bacteria and archaea reveals an extremely rapid and highly variable flux of genes in evolving microbial genomes, demonstrates that extensive gene loss and horizontal gene transfer leading to innovation are the two dominant evolutionary processes, and yields robust estimates of the supergenome size.

Adenoviral vector immunity: its implications and circumvention strategies.

Science.gov (United States)

Ahi, Yadvinder S; Bangari, Dinesh S; Mittal, Suresh K

2011-08-01

Adenoviral (Ad) vectors have emerged as a promising gene delivery platform for a variety of therapeutic and vaccine purposes during last two decades. However, the presence of preexisting Ad immunity and the rapid development of Ad vector immunity still pose significant challenges to the clinical use of these vectors. Innate inflammatory response following Ad vector administration may lead to systemic toxicity, drastically limit vector transduction efficiency and significantly abbreviate the duration of transgene expression. Currently, a number of approaches are being extensively pursued to overcome these drawbacks by strategies that target either the host or the Ad vector. In addition, significant progress has been made in the development of novel Ad vectors based on less prevalent human Ad serotypes and nonhuman Ad. This review provides an update on our current understanding of immune responses to Ad vectors and delineates various approaches for eluding Ad vector immunity. Approaches targeting the host and those targeting the vector are discussed in light of their promises and limitations.

Molecular design for recombinant adeno-associated virus (rAAV) vector production.

Science.gov (United States)

Aponte-Ubillus, Juan Jose; Barajas, Daniel; Peltier, Joseph; Bardliving, Cameron; Shamlou, Parviz; Gold, Daniel

2018-02-01

Recombinant adeno-associated virus (rAAV) vectors are increasingly popular tools for gene therapy applications. Their non-pathogenic status, low inflammatory potential, availability of viral serotypes with different tissue tropisms, and prospective long-lasting gene expression are important attributes that make rAAVs safe and efficient therapeutic options. Over the last three decades, several groups have engineered recombinant AAV-producing platforms, yielding high titers of transducing vector particles. Current specific productivity yields from different platforms range from 10 3 to 10 5 vector genomes (vg) per cell, and there is an ongoing effort to improve vector yields in order to satisfy high product demands required for clinical trials and future commercialization.Crucial aspects of vector production include the molecular design of the rAAV-producing host cell line along with the design of AAV genes, promoters, and regulatory elements. Appropriately, configuring and balancing the expression of these elements not only contributes toward high productivity, it also improves process robustness and product quality. In this mini-review, the rational design of rAAV-producing expression systems is discussed, with special attention to molecular strategies that contribute to high-yielding, biomanufacturing-amenable rAAV production processes. Details on molecular optimization from four rAAV expression systems are covered: adenovirus, herpesvirus, and baculovirus complementation systems, as well as a recently explored yeast expression system.

Adaptation of the short intergenic spacers between co-directional genes to the Shine-Dalgarno motif among prokaryote genomes

DEFF Research Database (Denmark)

Caro, Albert Pallejà; García-Vallvé, Santiago; Romeu, Antoni

2009-01-01

ABSTRACT: BACKGROUND: In prokaryote genomes most of the co-directional genes are in close proximity. Even the coding sequence or the stop codon of a gene can overlap with the Shine-Dalgarno (SD) sequence of the downstream co-directional gene. In this paper we analyze how the presence of SD may...... influence the stop codon usage or the spacing lengths between co-directional genes. RESULTS: The SD sequences for 530 prokaryote genomes have been predicted using computer calculations of the base-pairing free energy between translation initiation regions and the 16S rRNA 3' tail. Genomes with a large...... to the discussion of which factors affect the intergenic lengths, which cannot be totally explained by the pressure to compact the prokaryote genomes....

Adenoviral vector-mediated expression of B-50/GAP-43 induces alterations in the membrane organization of olfactory axon terminals in vivo

NARCIS (Netherlands)

Holtmaat, Anthony J D G; Hermens, W.T.J.M.C.; Sonnemans, M.A.F.; Giger, Roman J; Van Leeuwen, F W; Kaplitt, M G; Oestreicher, A B; Gispen, Willem Hendrik; Verhaagen, J

1997-01-01

B-50/GAP-43 is an intraneuronal membrane-associated growth cone protein with an important role in axonal growth and regeneration. By using adenoviral vector-directed expression of B-50/GAP-43 we studied the morphogenic action of B-50/GAP-43 in mature primary olfactory neurons that have established

[Sequences and expression pattern of mce gene in Leptospira interrogans of different serogroups].

Science.gov (United States)

Zhang, Lei; Xue, Feng; Yan, Jie; Mao, Ya-fei; Li, Li-wei

2008-11-01

To determine the frequency of mce gene in Leptospira interrogans, and to investigate the gene transcription levels of L. interrogans before and after infecting cells. The segments of entire mce genes from 13 L.interrogans strains and 1 L.biflexa strain were amplified by PCR and then sequenced after T-A cloning. A prokaryotic expression system of mce gene was constructed; the expression and output of the target recombinant protein rMce were examined by SDS-PAGE and Western Blot assay. Rabbits were intradermally immunized with rMce to prepare the antiserum, the titer of antiserum was measured by immunodiffusion test. The transcription levels of mce gene in L.interrogans serogroup Icterohaemorrhagiae serovar lai strain 56601 before and after infecting J774A.1 cells were monitored by real-time fluorescence quantitative RT-PCR. mce gene was carried in all tested L.interrogans strains, but not in L.biflexa serogroup Semaranga serovar patoc strain Patoc I. The similarities of nucleotide and putative amino acid sequences of the cloned mce genes to the reported sequences (GenBank accession No: NP712236) were 99.02%-100% and 97.91%-100%, respectively. The constructed prokaryotic expression system of mce gene expressed rMce and the output of rMce was about 5% of the total bacterial proteins. The antiserum against whole cell of L.interrogans strain 56601 efficiently recognized rMce. After infecting J774A.1 cells, transcription levels of the mce gene in L.interrogans strain 56601 were remarkably up-regulated. The constructed prokaryotic expression system of mce gene and the prepared antiserum against rMce provide useful tools for further study of the gene function.

Molecular cloning, structural analysis and expression of a zinc ...

African Journals Online (AJOL)

The results of prokaryotic expression of ZnBP and overexpression of the ZnBP gene in A. thaliana improve our understanding of the function of this gene. Future studies should investigate the molecular mechanisms involved in gland morphogenesis in cotton. Key words: Gossypium hirsutum, pigment gland, zinc binding ...

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

Science.gov (United States)

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

2009-01-01

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

Support vector machine for automatic pain recognition

Science.gov (United States)

Monwar, Md Maruf; Rezaei, Siamak

2009-02-01

Facial expressions are a key index of emotion and the interpretation of such expressions of emotion is critical to everyday social functioning. In this paper, we present an efficient video analysis technique for recognition of a specific expression, pain, from human faces. We employ an automatic face detector which detects face from the stored video frame using skin color modeling technique. For pain recognition, location and shape features of the detected faces are computed. These features are then used as inputs to a support vector machine (SVM) for classification. We compare the results with neural network based and eigenimage based automatic pain recognition systems. The experiment results indicate that using support vector machine as classifier can certainly improve the performance of automatic pain recognition system.

Novel strategy for generation and titration of recombinant adeno-associated virus vectors.

Science.gov (United States)

Shiau, Ai-Li; Liu, Pu-Ste; Wu, Chao-Liang

2005-01-01

Recombinant adeno-associated virus (rAAV) vectors have many advantages for gene therapeutic applications compared with other vector systems. Several methods that use plasmids or helper viruses have been reported for the generation of rAAV vectors. Unfortunately, the preparation of large-scale rAAV stocks is labor-intensive. Moreover, the biological titration of rAAV is still difficult, which may limit its preclinical and clinical applications. For this study, we developed a novel strategy to generate and biologically titrate rAAV vectors. A recombinant pseudorabies virus (PrV) with defects in its gD, gE, and thymidine kinase genes was engineered to express the AAV rep and cap genes, yielding PS virus, which served as a packaging and helper virus for the generation of rAAV vectors. PS virus was useful not only for generating high-titer rAAV vectors by cotransfection with an rAAV vector plasmid, but also for amplifying rAAV stocks. Notably, the biological titration of rAAV vectors was also feasible when cells were coinfected with rAAV and PS virus. Based on this strategy, we produced an rAAV that expresses prothymosin alpha (ProT). Expression of the ProT protein in vitro and in vivo mediated by rAAV/ProT gene transfer was detected by immunohistochemistry and a bioassay. Taken together, our results demonstrate that the PrV vector-based system is useful for generating rAAV vectors carrying various transgenes.

Polarization coupling of vector Bessel–Gaussian beams

International Nuclear Information System (INIS)

Takeuchi, Ryushi; Kozawa, Yuichi; Sato, Shunichi

2013-01-01

We report polarization coupling of radial and azimuthal electric field components of a vector light beam as predicted by the fact that the vector Helmholtz equation is expressed as coupled differential equations in cylindrical coordinates. To clearly observe the polarization variation of a beam as it propagates, higher order transverse modes of a vector Bessel–Gaussian beam were generated by a gain distribution modulation technique, which created a narrow ring-shaped gain region in a Nd:YVO 4 crystal. The polarization coupling was confirmed by the observation that the major polarization component of a vector Bessel–Gaussian beam alternates between radial and azimuthal components along with the propagation. (paper)

A Comprehensive Curation Shows the Dynamic Evolutionary Patterns of Prokaryotic CRISPRs

Directory of Open Access Journals (Sweden)

Guoqin Mai

2016-01-01

Full Text Available Motivation. Clustered regularly interspaced short palindromic repeat (CRISPR is a genetic element with active regulation roles for foreign invasive genes in the prokaryotic genomes and has been engineered to work with the CRISPR-associated sequence (Cas gene Cas9 as one of the modern genome editing technologies. Due to inconsistent definitions, the existing CRISPR detection programs seem to have missed some weak CRISPR signals. Results. This study manually curates all the currently annotated CRISPR elements in the prokaryotic genomes and proposes 95 updates to the annotations. A new definition is proposed to cover all the CRISPRs. The comprehensive comparison of CRISPR numbers on the taxonomic levels of both domains and genus shows high variations for closely related species even in the same genus. The detailed investigation of how CRISPRs are evolutionarily manipulated in the 8 completely sequenced species in the genus Thermoanaerobacter demonstrates that transposons act as a frequent tool for splitting long CRISPRs into shorter ones along a long evolutionary history.

On the uncertainty relations for vector-valued operators

International Nuclear Information System (INIS)

Chistyakov, A.L.

1976-01-01

In analogy with the expression for the Heisenberg incertainty principle in terms of dispersions by means of the Weyl inequality, in the case of one-dimensional quantum mechanical quantities, the principle for many-dimensional quantities can be expressed in terms of generalized dispersions and covariance matrices by means of inequalities similar to the Weyl unequality. The proofs of these inequalities are given in an abstract form, not only for the physical vector quantities, but also for arbitrary vector-valued operators with commuting self-adjoint components

Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection.

Science.gov (United States)

Trubitsyna, Maryia; Michlewski, Gracjan; Finnegan, David J; Elfick, Alistair; Rosser, Susan J; Richardson, Julia M; French, Christopher E

2017-06-02

Delivery of DNA to cells and its subsequent integration into the host genome is a fundamental task in molecular biology, biotechnology and gene therapy. Here we describe an IP-free one-step method that enables stable genome integration into either prokaryotic or eukaryotic cells. A synthetic mariner transposon is generated by flanking a DNA sequence with short inverted repeats. When purified recombinant Mos1 or Mboumar-9 transposase is co-transfected with transposon-containing plasmid DNA, it penetrates prokaryotic or eukaryotic cells and integrates the target DNA into the genome. In vivo integrations by purified transposase can be achieved by electroporation, chemical transfection or Lipofection of the transposase:DNA mixture, in contrast to other published transposon-based protocols which require electroporation or microinjection. As in other transposome systems, no helper plasmids are required since transposases are not expressed inside the host cells, thus leading to generation of stable cell lines. Since it does not require electroporation or microinjection, this tool has the potential to be applied for automated high-throughput creation of libraries of random integrants for purposes including gene knock-out libraries, screening for optimal integration positions or safe genome locations in different organisms, selection of the highest production of valuable compounds for biotechnology, and sequencing. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Challenging assumptions of notational transparency: the case of vectors in engineering mathematics

Science.gov (United States)

Craig, Tracy S.

2017-11-01

The notation for vector analysis has a contentious nineteenth century history, with many different notations describing the same or similar concepts competing for use. While the twentieth century has seen a great deal of unification in vector analysis notation, variation still remains. In this paper, the two primary notations used for expressing the components of a vector are discussed in historical and current context. Popular mathematical texts use the two notations as if they are transparent and interchangeable. In this research project, engineering students' proficiency at vector analysis was assessed and the data were analyzed using the Rasch measurement method. Results indicate that the students found items expressed in unit vector notation more difficult than those expressed in parenthesis notation. The expert experience of notation as transparent and unproblematically symbolic of underlying processes independent of notation is shown to contrast with the student experience where the less familiar notation is experienced as harder to work with.

pLIVE-EGFP: A liver specific vector carrying the EGFP reporter for ...

African Journals Online (AJOL)

-EGFP gene at the Xho I site of the pLIVE vector. The pLIVE-EGFP vector permits simultaneous expression of a gene of interest in addition to the EGFP reporter, specifically within liver cells, both in vivo and in vitro. When expressed in liver cells ...

II - Template Metaprogramming for Massively Parallel Scientific Computing - Vectorization with Expression Templates

CERN Multimedia

CERN. Geneva

2016-01-01

Large scale scientific computing raises questions on different levels ranging from the fomulation of the problems to the choice of the best algorithms and their implementation for a specific platform. There are similarities in these different topics that can be exploited by modern-style C++ template metaprogramming techniques to produce readable, maintainable and generic code. Traditional low-level code tend to be fast but platform-dependent, and it obfuscates the meaning of the algorithm. On the other hand, object-oriented approach is nice to read, but may come with an inherent performance penalty. These lectures aim to present he basics of the Expression Template (ET) idiom which allows us to keep the object-oriented approach without sacrificing performance. We will in particular show to to enhance ET to include SIMD vectorization. We will then introduce techniques for abstracting iteration, and introduce thread-level parallelism for use in heavy data-centric loads. We will show to to apply these methods i...

Adenoviral vectors for highly selective gene expression in central serotonergic neurons reveal quantal characteristics of serotonin release in the rat brain

Directory of Open Access Journals (Sweden)

Teschemacher Anja G

2009-03-01

Full Text Available Abstract Background 5-hydroxytryptamine (5 HT, serotonin is one of the key neuromodulators in mammalian brain, but many fundamental properties of serotonergic neurones and 5 HT release remain unknown. The objective of this study was to generate an adenoviral vector system for selective targeting of serotonergic neurones and apply it to study quantal characteristics of 5 HT release in the rat brain. Results We have generated adenoviral vectors which incorporate a 3.6 kb fragment of the rat tryptophan hydroxylase-2 (TPH-2 gene which selectively (97% co-localisation with TPH-2 target raphe serotonergic neurones. In order to enhance the level of expression a two-step transcriptional amplification strategy was employed. This allowed direct visualization of serotonergic neurones by EGFP fluorescence. Using these vectors we have performed initial characterization of EGFP-expressing serotonergic neurones in rat organotypic brain slice cultures. Fluorescent serotonergic neurones were identified and studied using patch clamp and confocal Ca2+ imaging and had features consistent with those previously reported using post-hoc identification approaches. Fine processes of serotonergic neurones could also be visualized in un-fixed tissue and morphometric analysis suggested two putative types of axonal varicosities. We used micro-amperometry to analyse the quantal characteristics of 5 HT release and found that central 5 HT exocytosis occurs predominantly in quanta of ~28000 molecules from varicosities and ~34000 molecules from cell bodies. In addition, in somata, we observed a minority of large release events discharging on average ~800000 molecules. Conclusion For the first time quantal release of 5 HT from somato-dendritic compartments and axonal varicosities in mammalian brain has been demonstrated directly and characterised. Release from somato-dendritic and axonal compartments might have different physiological functions. Novel vectors generated in this

Modulated molecular beam mass spectrometry: A generalized expression for the ''reaction product vector'' for linear systems

International Nuclear Information System (INIS)

Chang, H.; Weinberg, W.H.

1977-01-01

A generalized expression is developed that relates the ''reaction product vector'', epsilon exp(-iphi), to the kinetic parameters of a linear system. The formalism is appropriate for the analysis of modulated molecular beam mass spectrometry data and facilitates the correlation of experimental results to (proposed) linear models. A study of stability criteria appropriate for modulated molecular beam mass spectrometry experiments is also presented. This investigation has led to interesting inherent limitations which have not heretofore been emphasized, as well as a delineation of the conditions under which stable chemical oscillations may occur in the reacting system

An Update on Canine Adenovirus Type 2 and Its Vectors

Science.gov (United States)

Bru, Thierry; Salinas, Sara; Kremer, Eric J.

2010-01-01

Adenovirus vectors have significant potential for long- or short-term gene transfer. Preclinical and clinical studies using human derived adenoviruses (HAd) have demonstrated the feasibility of flexible hybrid vector designs, robust expression and induction of protective immunity. However, clinical use of HAd vectors can, under some conditions, be limited by pre-existing vector immunity. Pre-existing humoral and cellular anti-capsid immunity limits the efficacy and duration of transgene expression and is poorly circumvented by injections of larger doses and immuno-suppressing drugs. This review updates canine adenovirus serotype 2 (CAV-2, also known as CAdV-2) biology and gives an overview of the generation of early region 1 (E1)-deleted to helper-dependent (HD) CAV-2 vectors. We also summarize the essential characteristics concerning their interaction with the anti-HAd memory immune responses in humans, the preferential transduction of neurons, and its high level of retrograde axonal transport in the central and peripheral nervous system. CAV-2 vectors are particularly interesting tools to study the pathophysiology and potential treatment of neurodegenerative diseases, as anti-tumoral and anti-viral vaccines, tracer of synaptic junctions, oncolytic virus and as a platform to generate chimeric vectors. PMID:21994722

Prokaryotic Expression of Reindeer β-defensin-1 (reBD-1) Gene and Bioactivity Identification of the Recombinant Protein%驯鹿β-防御素-1(reBD-1)基因的原核表达及蛋白活性的鉴定

Institute of Scientific and Technical Information of China (English)

苏丽娜; 杨银凤; 景岚; 曹贵方

2011-01-01

The aim of this experiment was to construct prokaryotic expression vector of reindeer β-defensin-l(reBD-l), induce the expression of reBD-1 fusion protein in E. Coli, and evaluate the bioactivity of the expression products. The prepro-peptide of reBD-1 was amplified by RT-PCR. The mature peptide of encoding reBD-1 was amplified from recombinant cloning vector PMD19T/ reBD-1, and then cloned into pET-32a (+ ), in which reBD-1 fusion protein expression was induced by IPTG in E. Coli BL2KDE3). The expressed product was further cultured and purified for experiment of bacteriostasis in vitro. The results showed that the amplified products of prepro-peptide and mature peptide were 215 and 138 bp, respectively, and the homology of the sequences of the targeted gene and reBD-1 mRNA was up to 100%. The molecular weight of fusion proteins of prepro-peptide and mature peptide were 28 and 24 ku. The agar diffusion method has demonstrated that 0. 08 mg · mL-1 purified mature peptide protein has obvious antimicrobial activity against S. Aureus and E. Coli. From the results we can conclude that the prepro-peptide and mature peptides have high expression level in E. Coli, and the mature peptide has resistance to both Gram negative and Gram positive bacteria.%旨在构建驯鹿β防御素-1(Reindeerβ-defensin-1,reBD-1)基因的原核表达载体pET-32a(+ )/reBD-1,诱导reBD-1融合蛋白在大肠杆菌中表达,并对其表达产物的生物学活性进行评价.利用RT-PCR技术扩增reBD-1前原肽.从重组克隆载体PMD19T/reBD-1中扩增reBD-1成熟肽编码基因,并克隆入pET-32a(+)中,在大肠杆菌BL21 (DE3)中用IPTG诱导表达reBD-1融合蛋白.表达的融合蛋白扩大培养,进一步纯化后进行体外抑菌试验.结果表明,reBD-1前原肽和成熟肽扩增产物大小分别为215和138 bp,目的基因的序列与驯鹿防御素-1 mRNA序列同源性为100％.前原肽和成熟肽融合蛋白分子量分别为28和24 ku.利用琼脂糖扩散法表明,0

Deep sub-seafloor prokaryotes stimulated at interfaces over geological time RID B-1731-2010 RID A-1877-2008 RID D-2690-2009 RID A-2970-2010

DEFF Research Database (Denmark)

Parkes, RJ; Webster, G.; Cragg, BA

2005-01-01

in numbers of prokaryotes at depth were more restricted but also corresponded to increased activity; however, this time they were associated with repeating layers of diatom-rich sediments ( about 9 Myr old). These results show that deep sedimentary prokaryotes can have high activity, have changing diversity...... Pacific Ocean sites, margin and open ocean, both of which have deep, subsurface stimulation of prokaryotic processes associated with geochemical and/or sedimentary interfaces. At 90 m depth in the margin site, stimulation was such that prokaryote numbers were higher ( about 13-fold) and activity rates...

The Response of Heterotrophic Prokaryote and Viral Communities to Labile Organic Carbon Inputs Is Controlled by the Predator Food Chain Structure.

Science.gov (United States)

Sandaa, Ruth-Anne; Pree, Bernadette; Larsen, Aud; Våge, Selina; Töpper, Birte; Töpper, Joachim P; Thyrhaug, Runar; Thingstad, Tron Frede

2017-08-23

Factors controlling the community composition of marine heterotrophic prokaryotes include organic-C, mineral nutrients, predation, and viral lysis. Two mesocosm experiments, performed at an Arctic location and bottom-up manipulated with organic-C, had very different results in community composition for both prokaryotes and viruses. Previously, we showed how a simple mathematical model could reproduce food web level dynamics observed in these mesocosms, demonstrating strong top-down control through the predator chain from copepods via ciliates and heterotrophic nanoflagellates. Here, we use a steady-state analysis to connect ciliate biomass to bacterial carbon demand. This gives a coupling of top-down and bottom-up factors whereby low initial densities of ciliates are associated with mineral nutrient-limited heterotrophic prokaryotes that do not respond to external supply of labile organic-C. In contrast, high initial densities of ciliates give carbon-limited growth and high responsiveness to organic-C. The differences observed in ciliate abundance, and in prokaryote abundance and community composition in the two experiments were in accordance with these predictions. Responsiveness in the viral community followed a pattern similar to that of prokaryotes. Our study provides a unique link between the structure of the predator chain in the microbial food web and viral abundance and diversity.

PePPER : a webserver for prediction of prokaryote promoter elements and regulons

NARCIS (Netherlands)

de Jong, Anne; Pietersma, Hilco; Cordes, Martijn; Kuipers, Oscar P.; Kok, Jan

2012-01-01

Background: Accurate prediction of DNA motifs that are targets of RNA polymerases, sigma factors and transcription factors (TFs) in prokaryotes is a difficult mission mainly due to as yet undiscovered features in DNA sequences or structures in promoter regions. Improved prediction and comparison

Prokaryotic diversity of the Saccharomyces cerevisiae Atx1p-mediated copper pathway.

NARCIS (Netherlands)

Bakel, H. van; Huynen, M.A.; Wijmenga, C.

2004-01-01

MOTIVATION: Several genes involved in the cellular import of copper and its subsequent incorporation into the high-affinity iron transport complex in Saccharomyces cerevisiae are known to be conserved between eukaryotes and prokaryotes. However, the degree to which these genes share their functional

Impact of Lowland Rainforest Transformation on Diversity and Composition of Soil Prokaryotic Communities in Sumatra (Indonesia)

Science.gov (United States)

Schneider, Dominik; Engelhaupt, Martin; Allen, Kara; Kurniawan, Syahrul; Krashevska, Valentyna; Heinemann, Melanie; Nacke, Heiko; Wijayanti, Marini; Meryandini, Anja; Corre, Marife D.; Scheu, Stefan; Daniel, Rolf

2015-01-01

Prokaryotes are the most abundant and diverse group of microorganisms in soil and mediate virtually all biogeochemical cycles in terrestrial ecosystems. Thereby, they influence aboveground plant productivity and diversity. In this study, the impact of rainforest transformation to intensively managed cash crop systems on soil prokaryotic communities was investigated. The studied managed land use systems comprised rubber agroforests (jungle rubber), rubber plantations and oil palm plantations within two Indonesian landscapes Bukit Duabelas and Harapan. Soil prokaryotic community composition and diversity were assessed by pyrotag sequencing of bacterial and archaeal 16S rRNA genes. The curated dataset contained 16,413 bacterial and 1679 archaeal operational taxonomic units at species level (97% genetic identity). Analysis revealed changes in indigenous taxon-specific patterns of soil prokaryotic communities accompanying lowland rainforest transformation to jungle rubber, and intensively managed rubber and oil palm plantations. Distinct clustering of the rainforest soil communities indicated that these are different from the communities in the studied managed land use systems. The predominant bacterial taxa in all investigated soils were Acidobacteria, Actinobacteria, Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. Overall, the bacterial community shifted from proteobacterial groups in rainforest soils to Acidobacteria in managed soils. The archaeal soil communities were mainly represented by Thaumarchaeota and Euryarchaeota. Members of the Terrestrial Group and South African Gold Mine Group 1 (Thaumarchaeota) dominated in the rainforest and members of Thermoplasmata in the managed land use systems. The alpha and beta diversity of the soil prokaryotic communities was higher in managed land use systems than in rainforest. In the case of bacteria, this was related to soil characteristics such as pH value, exchangeable Ca and Fe content, C to N ratio

Impact of lowland rainforest transformation on diversity and composition of soil prokaryotic communities in Sumatra (Indonesia

Directory of Open Access Journals (Sweden)

Dominik eSchneider

2015-12-01

Full Text Available Prokaryotes are the most abundant and diverse group of microorganisms in soil and mediate virtually all biogeochemical cycles in terrestrial ecosystems. Thereby, they influence aboveground plant productivity and diversity. In this study, the impact of rainforest transformation to intensively managed cash crop systems on soil prokaryotic communities was investigated. The studied managed land use system comprised rubber agroforests (jungle rubber, rubber plantation and oil plantations within two Indonesian landscapes Bukit Duabelas and Harapan. Soil prokaryotic community composition and diversity were assessed by pyrotag sequencing of bacterial and archaeal 16S rRNA genes. The curated dataset contained 20,494 bacterial and 1,762 archaeal Operational Taxonomic Units at species level (97% genetic identity. Analysis revealed changes in indigenous taxon-specific patterns of soil prokaryotic communities accompanying lowland rainforest transformation to jungle rubber, and intensively managed rubber and oil palm plantations. Distinct clustering of the rainforest soil communities indicated that these are different from the communities in the studied managed land use systems. The predominant bacterial taxa in all investigated soils were Acidobacteria, Actinobacteria, Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. Overall, the bacterial community shifted from proteobacterial groups in rainforest soils to Acidobacteria in managed soils. The archaeal soil communities were mainly represented by Thaumarchaeota and Euryarchaeota. Members of the Terrestrial Group and South African Gold Mine Group 1 (Thaumarchaeota dominated in the rainforest and members of Thermoplasmata in the managed land use systems. The alpha and beta diversity of the soil prokaryotic communities was higher in managed land use systems than in rainforest. In the case of bacteria, this was related to soil characteristics such as pH value, exchangeable Ca and Fe content, C to

Prokaryotic Phylogenies Inferred from Whole-Genome Sequence and Annotation Data

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Wei Du

2013-01-01

Full Text Available Phylogenetic trees are used to represent the evolutionary relationship among various groups of species. In this paper, a novel method for inferring prokaryotic phylogenies using multiple genomic information is proposed. The method is called CGCPhy and based on the distance matrix of orthologous gene clusters between whole-genome pairs. CGCPhy comprises four main steps. First, orthologous genes are determined by sequence similarity, genomic function, and genomic structure information. Second, genes involving potential HGT events are eliminated, since such genes are considered to be the highly conserved genes across different species and the genes located on fragments with abnormal genome barcode. Third, we calculate the distance of the orthologous gene clusters between each genome pair in terms of the number of orthologous genes in conserved clusters. Finally, the neighbor-joining method is employed to construct phylogenetic trees across different species. CGCPhy has been examined on different datasets from 617 complete single-chromosome prokaryotic genomes and achieved applicative accuracies on different species sets in agreement with Bergey's taxonomy in quartet topologies. Simulation results show that CGCPhy achieves high average accuracy and has a low standard deviation on different datasets, so it has an applicative potential for phylogenetic analysis.

Prokaryotic community composition in alkaline-fermented skate (Raja pulchra).

Science.gov (United States)

Jang, Gwang Il; Kim, Gahee; Hwang, Chung Yeon; Cho, Byung Cheol

2017-02-01

Prokaryotes were extracted from skates and fermented skates purchased from fish markets and a local manufacturer in South Korea. The prokaryotic community composition of skates and fermented skates was investigated using 16S rRNA pyrosequencing. The ranges for pH and salinity of the grinded tissue extract from fermented skates were 8.4-8.9 and 1.6-6.6%, respectively. Urea and ammonia concentrations were markedly low and high, respectively, in fermented skates compared to skates. Species richness was increased in fermented skates compared to skates. Dominant and predominant bacterial groups present in the fermented skates belonged to the phylum Firmicutes, whereas those in skates belonged to Gammaproteobacteria. The major taxa found in Firmicutes were Atopostipes (Carnobacteriaceae, Lactobacillales) and/or Tissierella (Tissierellaceae, Tissierellales). A combination of RT-PCR and pyrosequencing for active bacterial composition showed that the dominant taxa i.e., Atopostipes and Tissierella, were active in fermented skate. Those dominant taxa are possibly marine lactic acid bacteria. Marine bacteria of the taxa Lactobacillales and/or Clostridia seem to be important in alkaline fermentation of skates. Copyright © 2016 Elsevier Ltd. All rights reserved.

Analysis of the promoters involved in enterocin AS-48 expression.

Science.gov (United States)

Cebrián, Rubén; Rodríguez-Ruano, Sonia; Martínez-Bueno, Manuel; Valdivia, Eva; Maqueda, Mercedes; Montalbán-López, Manuel

2014-01-01

The enterocin AS-48 is the best characterized antibacterial circular protein in prokaryotes. It is a hydrophobic and cationic bacteriocin, which is ribosomally synthesized by enterococcal cells and post-translationally cyclized by a head-to-tail peptide bond. The production of and immunity towards AS-48 depend upon the coordinated expression of ten genes organized in two operons, as-48ABC (where genes encoding enzymes with processing, secretion, and immunity functions are adjacent to the structural as-48A gene) and as-48C1DD1EFGH. The current study describes the identification of the promoters involved in AS-48 expression. Seven putative promoters have been here amplified, and separately inserted into the promoter-probe vector pTLR1, to create transcriptional fusions with the mCherry gene used as a reporter. The activity of these promoter regions was assessed measuring the expression of the fluorescent mCherry protein using the constitutive pneumococcal promoter PX as a reference. Our results revealed that only three promoters PA, P2(2) and PD1 were recognized in Enterococcus faecalis, Lactococcus lactis and Escherichia coli, in the conditions tested. The maximal fluorescence was obtained with PX in all the strains, followed by the P2(2) promoter, which level of fluorescence was 2-fold compared to PA and 4-fold compared to PD1. Analysis of putative factors influencing the promoter activity in single and double transformants in E. faecalis JH2-2 demonstrated that, in general, a better expression was achieved in presence of pAM401-81. In addition, the P2(2) promoter could be regulated in a negative fashion by genes existing in the native pMB-2 plasmid other than those of the as-48 cluster, while the pH seems to affect differently the as-48 promoter expression.

Analysis of the promoters involved in enterocin AS-48 expression.

Directory of Open Access Journals (Sweden)

Rubén Cebrián

Full Text Available The enterocin AS-48 is the best characterized antibacterial circular protein in prokaryotes. It is a hydrophobic and cationic bacteriocin, which is ribosomally synthesized by enterococcal cells and post-translationally cyclized by a head-to-tail peptide bond. The production of and immunity towards AS-48 depend upon the coordinated expression of ten genes organized in two operons, as-48ABC (where genes encoding enzymes with processing, secretion, and immunity functions are adjacent to the structural as-48A gene and as-48C1DD1EFGH. The current study describes the identification of the promoters involved in AS-48 expression. Seven putative promoters have been here amplified, and separately inserted into the promoter-probe vector pTLR1, to create transcriptional fusions with the mCherry gene used as a reporter. The activity of these promoter regions was assessed measuring the expression of the fluorescent mCherry protein using the constitutive pneumococcal promoter PX as a reference. Our results revealed that only three promoters PA, P2(2 and PD1 were recognized in Enterococcus faecalis, Lactococcus lactis and Escherichia coli, in the conditions tested. The maximal fluorescence was obtained with PX in all the strains, followed by the P2(2 promoter, which level of fluorescence was 2-fold compared to PA and 4-fold compared to PD1. Analysis of putative factors influencing the promoter activity in single and double transformants in E. faecalis JH2-2 demonstrated that, in general, a better expression was achieved in presence of pAM401-81. In addition, the P2(2 promoter could be regulated in a negative fashion by genes existing in the native pMB-2 plasmid other than those of the as-48 cluster, while the pH seems to affect differently the as-48 promoter expression.

A simple vector system to improve performance and utilisation of recombinant antibodies

Directory of Open Access Journals (Sweden)

Vincent Karen J

2006-12-01

Full Text Available Abstract Background Isolation of recombinant antibody fragments from antibody libraries is well established using technologies such as phage display. Phage display vectors are ideal for efficient display of antibody fragments on the surface of bacteriophage particles. However, they are often inefficient for expression of soluble antibody fragments, and sub-cloning of selected antibody populations into dedicated soluble antibody fragment expression vectors can enhance expression. Results We have developed a simple vector system for expression, dimerisation and detection of recombinant antibody fragments in the form of single chain Fvs (scFvs. Expression is driven by the T7 RNA polymerase promoter in conjunction with the inducible lysogen strain BL21 (DE3. The system is compatible with a simple auto-induction culture system for scFv production. As an alternative to periplasmic expression, expression directly in the cytoplasm of a mutant strain with a more oxidising cytoplasmic environment (Origami 2™ (DE3 was investigated and found to be inferior to periplasmic expression in BL21 (DE3 cells. The effect on yield and binding activity of fusing scFvs to the N terminus of maltose binding protein (a solubility enhancing partner, bacterial alkaline phosphatase (a naturally dimeric enzymatic reporter molecule, or the addition of a free C-terminal cysteine was determined. Fusion of scFvs to the N-terminus of maltose binding protein increased scFv yield but binding activity of the scFv was compromised. In contrast, fusion to the N-terminus of bacterial alkaline phosphatase led to an improved performance. Alkaline phosphatase provides a convenient tag allowing direct enzymatic detection of scFv fusions within crude extracts without the need for secondary reagents. Alkaline phosphatase also drives dimerisation of the scFv leading to an improvement in performance compared to monovalent constructs. This is illustrated by ELISA, western blot and

[Construction of eukaryotic recombinant vector and expression in COS7 cell of LipL32-HlyX fusion gene from Leptospira serovar Lai].

Science.gov (United States)

Huang, Bi; Bao, Lang; Zhong, Qi; Zhang, Huidong; Zhang, Ying

2009-04-01

This study was conducted to construct eukaryotic recombinant vector of LipL32-HlyX fusion gene from Leptospira serovar Lai and express it in mammalian cell. Both of LipL32 gene and HlyX gene were amplified from Leptospira strain O17 genomic DNA by PCR. Then with the two genes as template, LipL32-HlyX fusion gene was obtained by SOE PCR (gene splicing by overlap extension PCR). The fusion gene was then cloned into pcDNA3.1 by restriction nuclease digestion. Having been transformed into E. coli DH5alpha, the recombiant plasmid was identified by restriction nuclease digestion, PCR analysis and sequencing. The recombinant plasmid was then transfected into COS7 cell whose expression was detected by RT-PCR and Western blotting analysis. RT-PCR amplified a fragment about 2000 bp and Western blotting analysis found a specific band about 75 KD which was consistent with the expected fusion protein size. In conclusion, the successful construction of eukaryotic recombinant vector containing LipL32-HlyX fusion gene and the effective expression in mammalian have laid a foundation for the application of Leptospira DNA vaccine.

Matrix elements of a hyperbolic vector operator under SO(2,1)

International Nuclear Information System (INIS)

Zettili, N.; Boukahil, A.

2003-01-01

We deal here with the use of Wigner–Eckart type arguments to calculate the matrix elements of a hyperbolic vector operator V-vector by expressing them in terms of reduced matrix elements. In particular, we focus on calculating the matrix elements of this vector operator within the basis of the hyperbolic angular momentum T-vector whose components T-vector 1 , T-vector 2 , T-vector 3 satisfy an SO(2,1) Lie algebra. We show that the commutation rules between the components of V-vector and T-vector can be inferred from the algebra of ordinary angular momentum. We then show that, by analogy to the Wigner–Eckart theorem, we can calculate the matrix elements of V-vector within a representation where T-vector 2 and T-vector 3 are jointly diagonal. (author)

Stable suppression of myostatin gene expression in goat fetal fibroblast cells by lentiviral vector-mediated RNAi.

Science.gov (United States)

Patel, Utsav A; Patel, Amrutlal K; Joshi, Chaitanya G

2015-01-01

Myostatin (MSTN) is a secreted growth factor that negatively regulates skeletal muscle mass, and therefore, strategies to block myostatin-signaling pathway have been extensively pursued to increase the muscle mass in livestock. Here, we report a lentiviral vector-based delivery of shRNA to disrupt myostatin expression into goat fetal fibroblasts (GFFs) that were commonly used as karyoplast donors in somatic-cell nuclear transfer (SCNT) studies. Sh-RNA positive cells were screened by puromycin selection. Using real-time polymerase chain reaction (PCR), we demonstrated efficient knockdown of endogenous myostatin mRNA with 64% down-regulation in sh2 shRNA-treated GFF cells compared to GFF cells treated by control lentivirus without shRNA. Moreover, we have also demonstrated both the induction of interferon response and the expression of genes regulating myogenesis in GFF cells. The results indicate that myostatin-targeting siRNA produced endogenously could efficiently down-regulate myostatin expression. Therefore, targeted knockdown of the MSTN gene using lentivirus-mediated shRNA transgenics would facilitate customized cell engineering, allowing potential use in the establishment of stable cell lines to produce genetically engineered animals. © 2014 American Institute of Chemical Engineers.

Novel recombinant alphaviral and adenoviral vectors for cancer immunotherapy.

Science.gov (United States)

Osada, Takuya; Morse, Michael A; Hobeika, Amy; Lyerly, H Kim

2012-06-01

Although cellular immunotherapy based on autolgous dendritic cells (DCs) targeting antigens expressed by metastatic cancer has demonstrated clinical efficacy, the logistical challenges in generating an individualized cell product create an imperative to develop alternatives to DC-based cancer vaccines. Particularly attractive alternatives include in situ delivery of antigen and activation signals to resident antigen-presenting cells (APCs), which can be achieved by novel fusion molecules targeting the mannose receptor and by recombinant viral vectors expressing the antigen of interest and capable of infecting DCs. A particular challenge in the use of viral vectors is the well-appreciated clinical obstacles to their efficacy, specifically vector-specific neutralizing immune responses. Because heterologous prime and boost strategies have been demonstrated to be particularly potent, we developed two novel recombinant vectors based on alphaviral replicon particles and a next-generation adenovirus encoding an antigen commonly overexpressed in many human cancers, carcinoembryonic antigen (CEA). The rationale for developing these vectors, their unique characteristics, the preclinical studies and early clinical experience with each, and opportunities to enhance their effectiveness will be reviewed. The potential of each of these potent recombinant vectors to efficiently generate clinically active anti-tumor immune response alone, or in combination, will be discussed. Copyright © 2012 Elsevier Inc. All rights reserved.

CCR5 Gene Disruption via Lentiviral Vectors Expressing Cas9 and Single Guided RNA Renders Cells Resistant to HIV-1 Infection

Science.gov (United States)

Liu, Jingjing; Zhang, Di; Kimata, Jason T.; Zhou, Paul

2014-01-01

CCR5, a coreceptor for HIV-1 entry, is a major target for drug and genetic intervention against HIV-1. Genetic intervention strategies have knocked down CCR5 expression levels by shRNA or disrupted the CCR5 gene using zinc finger nucleases (ZFN) or Transcription activator-like effector nuclease (TALEN). In the present study, we silenced CCR5 via CRISPR associated protein 9 (Cas9) and single guided RNAs (sgRNAs). We constructed lentiviral vectors expressing Cas9 and CCR5 sgRNAs. We show that a single round transduction of lentiviral vectors expressing Cas9 and CCR5 sgRNAs into HIV-1 susceptible human CD4+ cells yields high frequencies of CCR5 gene disruption. CCR5 gene-disrupted cells are not only resistant to R5-tropic HIV-1, including transmitted/founder (T/F) HIV-1 isolates, but also have selective advantage over CCR5 gene-undisrupted cells during R5-tropic HIV-1 infection. Importantly, using T7 endonuclease I assay we did not detect genome mutations at potential off-target sites that are highly homologous to these CCR5 sgRNAs in stably transduced cells even at 84 days post transduction. Thus we conclude that silencing of CCR5 via Cas9 and CCR5-specific sgRNAs could be a viable alternative strategy for engineering resistance against HIV-1. PMID:25541967

Selection on start codons in prokaryotes and potential compensatory nucleotide substitutions.

Science.gov (United States)

Belinky, Frida; Rogozin, Igor B; Koonin, Eugene V

2017-09-29

Reconstruction of the evolution of start codons in 36 groups of closely related bacterial and archaeal genomes reveals purifying selection affecting AUG codons. The AUG starts are replaced by GUG and especially UUG significantly less frequently than expected under the neutral expectation derived from the frequencies of the respective nucleotide triplet substitutions in non-coding regions and in 4-fold degenerate sites. Thus, AUG is the optimal start codon that is actively maintained by purifying selection. However, purifying selection on start codons is significantly weaker than the selection on the same codons in coding sequences, although the switches between the codons result in conservative amino acid substitutions. The only exception is the AUG to UUG switch that is strongly selected against among start codons. Selection on start codons is most pronounced in evolutionarily conserved, highly expressed genes. Mutation of the start codon to a sub-optimal form (GUG or UUG) tends to be compensated by mutations in the Shine-Dalgarno sequence towards a stronger translation initiation signal. Together, all these findings indicate that in prokaryotes, translation start signals are subject to weak but significant selection for maximization of initiation rate and, consequently, protein production.

Manipulating the cell differentiation through lentiviral vectors.

Science.gov (United States)

Coppola, Valeria; Galli, Cesare; Musumeci, Maria; Bonci, Désirée

2010-01-01

The manipulation of cell differentiation is important to create new sources for the treatment of degenerative diseases or solve cell depletion after aggressive therapy against cancer. In this chapter, the use of a tissue-specific promoter lentiviral vector to obtain a myocardial pure lineage from murine embryonic stem cells (mES) is described in detail. Since the cardiac isoform of troponin I gene product is not expressed in skeletal or other muscle types, short mouse cardiac troponin proximal promoter is used to drive reporter genes. Cells are infected simultaneously with two lentiviral vectors, the first expressing EGFP to monitor the transduction efficiency, and the other expressing a puromycin resistance gene to select the specific cells of interest. This technical approach describes a method to obtain a pure cardiomyocyte population and can be applied to other lineages of interest.

Insulated piggyBac vectors for insect transgenesis

Directory of Open Access Journals (Sweden)

Horn Carsten

2006-06-01

Full Text Available Abstract Background Germ-line transformation of insects is now a widely used method for analyzing gene function and for the development of genetically modified strains suitable for pest control programs. The most widely used transposable element for the germ-line transformation of insects is piggyBac. The site of integration of the transgene can influence gene expression due to the effects of nearby transcription enhancers or silent heterochromatic regions. Position effects can be minimized by flanking a transgene with insulator elements. The scs/scs' and gypsy insulators from Drosophila melanogaster as well as the chicken β-globin HS4 insulator function in both Drosophila and mammalian cells. Results To minimize position effects we have created a set of piggyBac transformation vectors that contain either the scs/scs', gypsy or chicken β-globin HS4 insulators. The vectors contain either fluorescent protein or eye color marker genes and have been successfully used for germ-line transformation of Drosophila melanogaster. A set of the scs/scs' vectors contains the coral reef fluorescent protein marker genes AmCyan, ZsGreen and DsRed that have not been optimized for translation in human cells. These marker genes are controlled by a combined GMR-3xP3 enhancer/promoter that gives particularly strong expression in the eyes. This is also the first report of the use of the ZsGreen and AmCyan reef fluorescent proteins as transformation markers in insects. Conclusion The insulated piggyBac vectors should protect transgenes against position effects and thus facilitate fine control of gene expression in a wide spectrum of insect species. These vectors may also be used for transgenesis in other invertebrate species.

Viruses and Protists Induced-mortality of Prokaryotes around the Antarctic Peninsula during the Austral Summer

KAUST Repository

Vaque, Dolors; Boras, Julia A.; Torrent-Llagostera, Francesc; Agusti, Susana; Arrieta, J M; Lara, Elena; Castillo, Yaiza M.; Duarte, Carlos M.; Sala, Maria M.

2017-01-01

During the Austral summer 2009 we studied three areas surrounding the Antarctic Peninsula: the Bellingshausen Sea, the Bransfield Strait and the Weddell Sea. We aimed to investigate, whether viruses or protists were the main agents inducing prokaryotic mortality rates, and the sensitivity to temperature of prokaryotic heterotrophic production and mortality based on the activation energy (Ea) for each process. Seawater samples were taken at seven depths (0.1-100 m) to quantify viruses, prokaryotes and protists abundances, and heterotrophic prokaryotic production (PHP). Viral lytic production, lysogeny, and mortality rates of prokaryotes due to viruses and protists were estimated at surface (0.1-1 m) and at the Deep Fluorescence Maximum (DFM, 12-55 m) at eight representative stations of the three areas. The average viral lytic production ranged from 1.0 +/- 0.3 x 10(7) viruses ml(-1) d(-1) in the Bellingshausen Sea to1.3 +/- 0.7 x 10(7) viruses ml(-1) d(-1) in the Bransfield Strait, while lysogeny, when detectable, recorded the lowest value in the Bellingshausen Sea (0.05 +/- 0.05 x 10(7) viruses ml(-1) d(-1)) and the highest in the Weddell Sea (4.3 +/- 3.5 x 10(7) viruses ml(-1) d(-1)). Average mortality rates due to viruses ranged from 9.7 +/- 6.1 x 10(4) cells ml(-1) d(-1) in the Weddell Sea to 14.3 +/- 4.0 x 10(4) cells ml(-1) d(-1) in the Bellingshausen Sea, and were higher than averaged grazing rates in the Weddell Sea (5.9 +/- 1.1 x 10(4) cells ml(-1) d(-1)) and in the Bellingshausen Sea (6.8 +/- 0.9 x 10(4) cells ml-1 d(-1)). The highest impact on prokaryotes by viruses and main differences between viral and protists activities were observed in surface samples: 17.8 +/- 6.8 x 10(4) cells ml(-1) d(-1) and 6.5 +/- 3.9 x 10(4) cells ml(-1) d(-1) in the Weddell Sea; 22.1 +/- 9.6 x 10(4) cells ml(-1) d(-1) and 11.6 +/- 1.4 x 10(4) cells ml(-1) d(-1) in the Bransfield Strait; and 16.1 +/- 5.7 x 10(4) cells ml(-1) d(-1) and 7.9 +/- 2.6 x 10(4) cells ml(-1) d(-1) in

Viruses and Protists Induced-mortality of Prokaryotes around the Antarctic Peninsula during the Austral Summer

KAUST Repository

Vaque, Dolors

2017-03-27

During the Austral summer 2009 we studied three areas surrounding the Antarctic Peninsula: the Bellingshausen Sea, the Bransfield Strait and the Weddell Sea. We aimed to investigate, whether viruses or protists were the main agents inducing prokaryotic mortality rates, and the sensitivity to temperature of prokaryotic heterotrophic production and mortality based on the activation energy (Ea) for each process. Seawater samples were taken at seven depths (0.1-100 m) to quantify viruses, prokaryotes and protists abundances, and heterotrophic prokaryotic production (PHP). Viral lytic production, lysogeny, and mortality rates of prokaryotes due to viruses and protists were estimated at surface (0.1-1 m) and at the Deep Fluorescence Maximum (DFM, 12-55 m) at eight representative stations of the three areas. The average viral lytic production ranged from 1.0 +/- 0.3 x 10(7) viruses ml(-1) d(-1) in the Bellingshausen Sea to1.3 +/- 0.7 x 10(7) viruses ml(-1) d(-1) in the Bransfield Strait, while lysogeny, when detectable, recorded the lowest value in the Bellingshausen Sea (0.05 +/- 0.05 x 10(7) viruses ml(-1) d(-1)) and the highest in the Weddell Sea (4.3 +/- 3.5 x 10(7) viruses ml(-1) d(-1)). Average mortality rates due to viruses ranged from 9.7 +/- 6.1 x 10(4) cells ml(-1) d(-1) in the Weddell Sea to 14.3 +/- 4.0 x 10(4) cells ml(-1) d(-1) in the Bellingshausen Sea, and were higher than averaged grazing rates in the Weddell Sea (5.9 +/- 1.1 x 10(4) cells ml(-1) d(-1)) and in the Bellingshausen Sea (6.8 +/- 0.9 x 10(4) cells ml-1 d(-1)). The highest impact on prokaryotes by viruses and main differences between viral and protists activities were observed in surface samples: 17.8 +/- 6.8 x 10(4) cells ml(-1) d(-1) and 6.5 +/- 3.9 x 10(4) cells ml(-1) d(-1) in the Weddell Sea; 22.1 +/- 9.6 x 10(4) cells ml(-1) d(-1) and 11.6 +/- 1.4 x 10(4) cells ml(-1) d(-1) in the Bransfield Strait; and 16.1 +/- 5.7 x 10(4) cells ml(-1) d(-1) and 7.9 +/- 2.6 x 10(4) cells ml(-1) d(-1) in

The 3D protein of duck hepatitis A virus type 1 binds to a viral genomic 3' UTR and shows RNA-dependent RNA polymerase activity.

Science.gov (United States)

Zhang, Yu; Cao, Qianda; Wang, Mingshu; Jia, Renyong; Chen, Shun; Zhu, Dekang; Liu, Mafeng; Sun, Kunfeng; Yang, Qiao; Wu, Ying; Zhao, Xinxin; Chen, Xiaoyue; Cheng, Anchun

2017-12-01

To explore the RNA-dependent RNA polymerase (RdRP) function of the 3D protein of duck hepatitis A virus type 1 (DHAV-1), the gene was cloned into the pET-32a(+) vector for prokaryotic expression. The 3' untranslated region (3' UTR) of DHAV-1 together with a T7 promoter was cloned into the pMD19-T vector for in vitro transcription of 3' UTR RNA, which was further used as a template in RNA-dependent RNA polymerization. In this study, three methods were applied to analyze the RdRP function of the 3D protein: (1) ammonium molybdate spectrophotometry to detect pyrophosphate produced during polymerization; (2) quantitative reverse transcription PCR (RT-qPCR) to investigate the changes in RNA quantity during polymerization; and (3) electrophoresis mobility shift assay to examine the interaction between the 3D protein and 3' UTR. The results showed the 3D protein was successfully expressed in bacteria culture supernatant in a soluble form, which could be purified by affinity chromatography. In 3D enzymatic activity assays, pyrophosphate and RNA were produced, the amounts of which increased based on approximative kinetics, and binding of the 3D protein to the 3' UTR was observed. These results indicate that prokaryotically expressed soluble DHAV-13D protein can bind to a viral genomic 3' UTR and exhibit RdRP activity.

Footprints of Optimal Protein Assembly Strategies in the Operonic Structure of Prokaryotes

Directory of Open Access Journals (Sweden)

Jan Ewald

2015-04-01

Full Text Available In this work, we investigate optimality principles behind synthesis strategies for protein complexes using a dynamic optimization approach. We show that the cellular capacity of protein synthesis has a strong influence on optimal synthesis strategies reaching from a simultaneous to a sequential synthesis of the subunits of a protein complex. Sequential synthesis is preferred if protein synthesis is strongly limited, whereas a simultaneous synthesis is optimal in situations with a high protein synthesis capacity. We confirm the predictions of our optimization approach through the analysis of the operonic organization of protein complexes in several hundred prokaryotes. Thereby, we are able to show that cellular protein synthesis capacity is a driving force in the dissolution of operons comprising the subunits of a protein complex. Thus, we also provide a tested hypothesis explaining why the subunits of many prokaryotic protein complexes are distributed across several operons despite the presumably less precise co-regulation.

Transient expression of the influenza A virus PB1-F2 protein using a plum pox virus-based vector in Nicotiana benthamiana.

Science.gov (United States)

Kamencayová, M; Košík, I; Hunková, J; Subr, Z W

2014-01-01

PB1-F2 protein of influenza A virus (IAV) was cloned in a plum pox virus (PPV) genome-based vector and attempts to express it in biolistically transfected Nicotiana benthamiana plants were performed. The vector-insert construct replicated in infected plants properly and was stable during repeated passage by mechanical inoculation, as demonstrated by disease symptoms and immunoblot detection of PPV capsid protein, while PB1-F2-specific band was more faint. We showed that it was due its low solubility. Modification of sample preparation (denaturation/solubilization preceding the centrifugation of cell debris) led to substantial signal enhancement. Maximal level of PB1-F2 expression in plants was observed 12 days post inoculation (dpi). Only 1% SDS properly solubilized the protein, other detergents were much less efficient. Solubilization with 8M urea released approximately 50% of PB1-F2 from the plant tissues, thus the treatment with this removable chaotropic agent may be a good starting point for the purification of the protein for eventual functional studies in the future.

Cloning and expression of chaetomium thermophilum xylanase 11-A gene in prokaryote

International Nuclear Information System (INIS)

Wajid, S.; Latif, F.; Afzal, S.; Rajoka, I.

2008-01-01

The xylanase gene was cloned into pET32a(+) and expressed in E. coli BL21 under T7 promotor alongwith fusion protein. The SDS-PAGE and western blot analysis showed a protein of 42 kDa. The best expression of xylanase enzyme was found by using xylose as carbon source and lactose as an inducer. The maximum activity of xylanase expressed in E. coli was 6.02 U/mL in the presence of 2% xylose in DS medium. The activity of recombinant xylanase was observed on 1% xylan LB agar plates, showed halos of xylan clearance when lactose was used as an inducer. (author)

Hypoxia- and radiation-inducible, breast cell-specific targeting of retroviral vectors

International Nuclear Information System (INIS)

Lipnik, Karoline; Greco, Olga; Scott, Simon; Knapp, Elzbieta; Mayrhofer, Elisabeth; Rosenfellner, Doris; Guenzburg, Walter H.; Salmons, Brian; Hohenadl, Christine

2006-01-01

To facilitate a more efficient radiation and chemotherapy of mammary tumours, synthetic enhancer elements responsive to hypoxia and ionizing radiation were coupled to the mammary-specific minimal promoter of the murine whey acidic protein (WAP) encoding gene. The modified WAP promoter was introduced into a retroviral promoter conversion (ProCon) vector. Expression of a transduced reporter gene in response to hypoxia and radiation was analysed in stably infected mammary cancer cell lines and an up to 9-fold increase in gene expression demonstrated in comparison to the respective basic vector. Expression analyses in vitro, moreover, demonstrated a widely preserved mammary cell-specific promoter activity. For in vivo analyses, xenograft tumours consisting of infected human mammary adenocarcinoma cells were established in SCID/beige mice. Immunohistochemical analyses demonstrated a hypoxia-specific, markedly increased WAP promoter-driven expression in these tumours. Thus, this retroviral vector will facilitate a targeted gene therapeutic approach exploiting the unique environmental condition in solid tumours