scholarly journals A series of constitutive expression vectors to accurately measure the rate of DNA transposition and correct for auto-inhibition

2018 ◽  
Author(s):  
Michael Tellier ◽  
Ronald Chalmers
Keyword(s):  
Dna Sequences ◽  
Constitutive Expression ◽  
Expression Vectors ◽  
Single Chain ◽  
Dimer Interface ◽  
Inducible Promoters ◽  
Wide Range ◽  
Genomic Damage ◽  
Screening Assays ◽  
Transposase Expression

AbstractBackgroundTransposable elements (TEs) form a diverse group of DNA sequences encoding functions for their own mobility. This ability has been exploited as a powerful tool for molecular biology and genomics techniques. However, their use is sometimes limited because their activity is auto-regulated to allow them to cohabit within their hosts without causing excessive genomic damage. To overcome these limitations, it is important to develop efficient and simple screening assays for hyperactive transposases.ResultsTo widen the range of transposase expression normally accessible with inducible promoters, we have constructed a set of vectors based on constitutive promoters of different strengths. We characterized and validated our expression vectors with Hsmar1, a member of the mariner transposon family. We observed the highest rate of transposition with the weakest promoters. We went on to investigate the effects of mutations in the Hsmar1 transposase dimer interface and of covalently linking two transposase monomers in a single-chain dimer. We also tested the severity of mutations in the lineage leading to the human SETMAR gene, in which one copy of the Hsmar1 transposase has contributed a domain.ConclusionsWe generated a set of vectors to provide a wide range of transposase expression which will be useful for screening libraries of transposase mutants. We also found that mutations in the Hsmar1 dimer interface provides resistance to overproduction inhibition in bacteria, which could be valuable for improving bacterial transposon mutagenesis techniques.

scholarly journals Compensating for over-production inhibition of the Hsmar1 transposon in Escherichia coli using a series of constitutive promoters

Mobile DNA ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Michael Tellier ◽  
Ronald Chalmers
Keyword(s):  
Escherichia Coli ◽  
Essential Gene ◽  
Point Mutations ◽  
Regulatory Mechanisms ◽  
Expression Vectors ◽  
White Background ◽  
Single Chain ◽  
Host Chromosome ◽  
Wide Range ◽  
Transposase Expression

Abstract Background Transposable elements (TEs) are a diverse group of self-mobilizing DNA elements. Transposition has been exploited as a powerful tool for molecular biology and genomics. However, transposition is sometimes limited because of auto-regulatory mechanisms that presumably allow them to cohabit within their hosts without causing excessive genomic damage. The papillation assay provides a powerful visual screen for hyperactive transposases. Transposition is revealed by the activation of a promoter-less lacZ gene when the transposon integrates into a non-essential gene on the host chromosome. Transposition events are detected as small blue speckles, or papillae, on the white background of the main Escherichia coli colony. Results We analysed the parameters of the papillation assay including the strength of the transposase transcriptional and translational signals. To overcome certain limitations of inducible promoters, we constructed a set of vectors based on constitutive promoters of different strengths to widen the range of transposase expression. We characterized and validated our expression vectors with Hsmar1, a member of the mariner transposon family. The highest rate of transposition was observed with the weakest promoters. We then took advantage of our approach to investigate how the level of transposition responds to selected point mutations and the effect of joining the transposase monomers into a single-chain dimer. Conclusions We generated a set of vectors to provide a wide range of transposase expression which will be useful for screening libraries of transposase mutants. The use of weak promoters should allow screening for truly hyperactive transposases rather than those that are simply resistant to auto-regulatory mechanisms, such as overproduction inhibition (OPI). We also found that mutations in the Hsmar1 dimer interface provide resistance to OPI in bacteria, which could be valuable for improving bacterial transposon mutagenesis techniques.

scholarly journals A Productive Expression Platform Derived from Host-Restricted Eilat Virus: Its Extensive Validation and Novel Strategy

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 660
Author(s):  
Lu Tan ◽  
Yiwen Zhang ◽  
Xingxing Wang ◽  
Dal Young Kim
Keyword(s):  
Expression Vectors ◽  
Structural Genes ◽  
Host Restriction ◽  
Expression Platform ◽  
Mosquito Cells ◽  
Wide Range ◽  
Restriction Property ◽  
Infected Cells ◽  
Multiple Levels ◽  
Novel Strategy

Most alphaviruses are transmitted by mosquitoes and infect a wide range of insects and vertebrates. However, Eilat virus (EILV) is defective for infecting vertebrate cells at multiple levels of the viral life cycle. This host-restriction property renders EILV an attractive expression platform since it is not infectious for vertebrates and therefore provides a highly advantageous safety profile. Here, we investigated the feasibility of versatile EILV-based expression vectors. By replacing the structural genes of EILV with those of other alphaviruses, we generated seven different chimeras. These chimeras were readily rescued in the original mosquito cells and were able to reach high titers, suggesting that EILV is capable of packaging the structural proteins of different lineages. We also explored the ability of EILV to express authentic antigens via double subgenomic (SG) RNA vectors. Four foreign genetic materials of varied length were introduced into the EILV genome, and the expressed heterologous genetic materials were readily detected in the infected cells. By inserting an additional SG promoter into the chimera genome containing the structural genes of Chikungunya virus (CHIKV), we developed a bivalent vaccine candidate against CHIKV and Zika virus. These data demonstrate the outstanding compatibility of the EILV genome. The produced recombinants can be applied to vaccine and diagnostic tool development, but more investigations are required.

Regulated overproduction of the GAL4 gene product greatly increases expression from galactose-inducible promoters on multi-copy expression vectors in yeast

Gene ◽  
1987 ◽  
Vol 61 (2) ◽  
pp. 123-133 ◽  
Author(s):  
Loren D. Schultz ◽  
Kathryn J. Hofmann ◽  
Lawrence M. Mylin ◽  
Donna L. Montgomery ◽  
Ronald W. Ellis ◽  
...  
Keyword(s):  
Gene Product ◽  
Expression Vectors ◽  
Inducible Promoters

scholarly journals A New Statistic for Detecting Genetic Differentiation

Genetics ◽  
2000 ◽  
Vol 155 (4) ◽  
pp. 2011-2014 ◽  
Author(s):  
Richard R Hudson
Keyword(s):  
Genetic Differentiation ◽  
Dna Sequences ◽  
Genetic Data ◽  
Island Model ◽  
Wide Range ◽  
Infinite Sites Model ◽  
Parameter Values

Abstract A new statistic for detecting genetic differentiation of subpopulations is described. The statistic can be calculated when genetic data are collected on individuals sampled from two or more localities. It is assumed that haplotypic data are obtained, either in the form of DNA sequences or data on many tightly linked markers. Using a symmetric island model, and assuming an infinite-sites model of mutation, it is found that the new statistic is as powerful or more powerful than previously proposed statistics for a wide range of parameter values.

Parasites in Antarctic krill guts inferred from DNA sequences

2019 ◽  
Vol 31 (1) ◽  
pp. 16-22 ◽  
Author(s):  
Alison C. Cleary ◽  
Maria C. Casas ◽  
Edward G. Durbin ◽  
Jaime Gómez-Gutiérrez
Keyword(s):  
Dna Sequences ◽  
High Frequency ◽  
Dna Analysis ◽  
Antarctic Krill ◽  
Euphausia Superba ◽  
Gut Contents ◽  
The West ◽  
West Antarctic ◽  
Wide Range

AbstractThe keystone role of Antarctic krill,Euphausia superbaDana, in Southern Ocean ecosystems, means it is essential to understand the factors controlling their abundance and secondary production. One such factor that remains poorly known is the role of parasites. A recent study of krill diet using DNA analysis of gut contents provided a snapshot of the parasites present within 170E. superbaguts in a small area along the West Antarctic Peninsula. These parasites includedMetschnikowiaspp. fungi,Haptoglossasp. peronosporomycetes,LankesteriaandParalecudinaspp. apicomplexa,Stegophorussp. nematodes, andPseudocolliniaspp. ciliates. Of these parasites,Metschnikowiaspp. fungi andPseudocolliniaspp. ciliates had previously been observed inE. superba, as had other genera of apicomplexans, though notLankesteriaandParalecudina.In contrast, nematodes had previously only been observed in eggs ofE. superba, and there are no literature reports of peronosporomycetes in euphausiids.Pseudocolliniaspp., parasitoids which obligately kill their host, were the most frequently observed infection, with a prevalence of 12%. The wide range of observed parasites and the relatively high frequency of infections suggest parasites may play a more important role than previously acknowledged inE. superbaecology and population dynamics.

Species-specific duplex PCR for the detection of Pratylenchus penetrans

Nematology ◽  
2009 ◽  
Vol 11 (6) ◽  
pp. 847-857 ◽  
Author(s):  
Lieven Waeyenberge ◽  
Nicole Viaene ◽  
Maurice Moens
Keyword(s):  
Internal Control ◽  
Dna Sequences ◽  
Rrna Gene ◽  
Duplex Pcr ◽  
Specific Primers ◽  
5.8S Rrna Gene ◽  
5.8S Rrna ◽  
Wide Range ◽  
Pcr Products ◽  
Species Specific

Abstract ITS1, the 5.8S rRNA gene and ITS2 of the rDNA region were sequenced from 20 different Pratylenchus species. Additionally, the same region was sequenced from seven populations of P. penetrans. After purifying, cloning and sequencing the PCR products, all sequences were aligned in order to find unique sites suitable for the design of species-specific primers for P. penetrans. Since ITS regions showed variability between and even within populations of P. penetrans, only three small DNA sequences were suitable for the construction of three potentially useful species-specific primers. New species-specific primers were paired with existing universal ITS primers and tested in all possible primer combinations. The best performing primer set, supplemented with a universal 28S rDNA primer set that served as an internal control, was tested in duplex PCR. The ideal annealing temperature, Mg2+ concentration and primer ratios were then determined for the most promising primer set. The optimised duplex PCR was subsequently tested on a wide range of different Pratylenchus spp. and 25 P. penetrans populations originating from all over the world. To test the sensitivity, the duplex PCR was conducted on DNA extracted from a single P. penetrans nematode mixed with varying amounts of nematodes belonging to another Pratylenchus species. Results showed that a reliable and sensitive P. penetrans species-specific duplex PCR was constructed.

scholarly journals New Recombinant Mycobacterium bovis BCG Expression Vectors: Improving Genetic Control over Mycobacterial Promoters

2016 ◽  
Vol 82 (8) ◽  
pp. 2240-2246 ◽  
Author(s):  
Alex I. Kanno ◽  
Cibelly Goulart ◽  
Henrique K. Rofatto ◽  
Sergio C. Oliveira ◽  
Luciana C. C. Leite ◽  
...  
Keyword(s):  
Mycobacterium Bovis ◽  
Fluorescent Protein ◽  
Vaccine Development ◽  
Expression Vectors ◽  
Content Type ◽  
Expression Levels ◽  
Wide Range ◽  
Mycobacteriophage L5 ◽  
Green Fluorescent ◽  
Selection Of

ABSTRACTThe expression of many antigens, stimulatory molecules, or even metabolic pathways in mycobacteria such asMycobacterium bovisBCG orM. smegmatiswas made possible through the development of shuttle vectors, and several recombinant vaccines have been constructed. However, gene expression in any of these systems relied mostly on the selection of natural promoters expected to provide the required level of expression by trial and error. To establish a systematic selection of promoters with a range of strengths, we generated a library of mutagenized promoters through error-prone PCR of the strong PL5promoter, originally from mycobacteriophage L5. These promoters were cloned upstream of the enhanced green fluorescent protein reporter gene, and recombinantM. smegmatisbacteria exhibiting a wide range of fluorescence levels were identified. A set of promoters was selected and identified as having high (pJK-F8), intermediate (pJK-B7, pJK-E6, pJK-D6), or low (pJK-C1) promoter strengths in bothM. smegmatisandM. bovisBCG. The sequencing of the promoter region demonstrated that it was extensively modified (6 to 11%) in all of the plasmids selected. To test the functionality of the system, two different expression vectors were demonstrated to allow corresponding expression levels of theSchistosoma mansoniantigen Sm29 in BCG. The approach used here can be used to adjust expression levels for synthetic and/or systems biology studies or for vaccine development to maximize the immune response.

Progression of the transformed phenotype in clonal lines of Abelson virus-infected lymphocytes

1983 ◽  
Vol 3 (4) ◽  
pp. 596-604
Author(s):  
C A Whitlock ◽  
S F Ziegler ◽  
O N Witte
Keyword(s):  
Cell Lines ◽  
Dna Sequences ◽  
Lymphoid Cells ◽  
Growth Potential ◽  
Malignant Growth ◽  
Wide Range ◽  
Feeder Layers ◽  
Growth Phenotype ◽  
Abelson Virus

Some molecular changes which correlate with the tumorigenic progression of neoplastic cells can best be studied with in vitro cell lines that represent each stage in the progression. Lymphoid cells infected by Abelson murine leukemia virus exhibit a wide range of growth potential in vitro and in vivo. Uncloned populations that are poorly oncogenic early after infection become progressively more oncogenic with successive passages of the cells in culture. In such mass cultures, it is difficult to evaluate whether a rare subpopulation of highly oncogenic cells becomes dominant in the culture or whether the individual cells progress in oncogenic phenotype. To examine this latter possibility, Abelson virus-infected lymphoid cells were cloned by limiting-dilution culture 10 days postinfection. We isolated two clones that grew poorly in agar, required feeder layers of adherent bone marrow cells for growth in liquid culture, and were extremely slow to form tumors in syngeneic animals. Both clones, after passage in the presence of adherent feeder layers for 3 months, grew well in liquid and agar-containing cultures in the absence of feeder layers and formed tumors in animals at a rapid rate. The progression of these clonal cell lines to a more malignant growth phenotype occurred in the absence of detectable changes in the concentration, half-life, phosphorylation, in vitro kinase activity, or cell localization of the Abelson virus-encoded transforming protein. No change in the concentration or arrangement of integrated Abelson viral DNA sequences was detected in either clone. Thus, perhaps changes in the expression of cellular genes would appear to alter the growth properties of lymphoid cells after their initial transformation by Abelson virus. Such cellular changes could complement the activity of the Abelson virus transforming protein in producing the fully malignant growth phenotype.

scholarly journals Characterization of Monoclonal Antibodies against Bovine herpesvirus type 1 selected by Phage Display Technology

2017 ◽  
Vol 38 (6) ◽  
pp. 3915
Author(s):  
Greice Japolla ◽  
Ana Flávia Batista Penido ◽  
Greyciele Rodrigues Almeida ◽  
Luiz Artur Mendes Bataus ◽  
Jair Pereira Cunha Junior ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Phage Display ◽  
Bovine Herpesvirus ◽  
Single Chain ◽  
Phage Display Technology ◽  
Herpesvirus Type ◽  
Wide Range ◽  
Display Technology ◽  
Bovine Herpesvirus Type 1

The specificity of monoclonal antibodies (mAbs) to desired targets makes these molecules suitable for therapeutic and diagnostic uses against a wide range of pathogens. Phage display antibody libraries offer one method by which mAbs can be selected for, without the use of conventional hybridoma technology. In this work, phage display technology was used to construct, select and characterize a combinatorial single chain fragment variable (scFv) antibody library against bovine herpesvirus type 1 (BoHV-1) from the immune repertoire of chickens immunized with the virus. In silico analysis of the hypervariable domains of the antibody heavy chains revealed a high frequency of scFv fragments with low variability, suggesting that selection had probably been carried out and favored by a few im-munogenic viral antigens. The reactivity of the scFv fragments selected against BoHV-1 was demon-strated by Phage-ELISA. A significant increase in antibody reactivity to the target was observed after six rounds of library selection, showing its potential use as a molecule for BoHV-1 diagnosis. The strategy described here opens up a field for the use of phage display as a tool for selection of mono-clonal antibodies that could be used for theranostic applications against infectious and parasitic dis-eases of veterinary interest.

scholarly journals Constitutive expression of bcl-2 in B cells causes a lethal form of lupuslike autoimmune disease after induction of neonatal tolerance to H-2b alloantigens.

1996 ◽  
Vol 183 (6) ◽  
pp. 2523-2531 ◽  
Author(s):  
M López-Hoyos ◽  
R Carrió ◽  
R Merino ◽  
L Buelta ◽  
S Izui ◽  
...  
Keyword(s):  
B Cells ◽  
Autoimmune Disease ◽  
Constitutive Expression ◽  
Systemic Autoimmune Disease ◽  
Spleen Cells ◽  
Term Survival ◽  
F1 Hybrid ◽  
Wide Range

The bcl-2 protooncogene has been shown to provide a survival signal to self-reactive B cells, but it fails to override their developmental arrest after encounter with antigen. Furthermore, constitutive expression of bcl-2 in B cells does not promote the development of autoimmune disease in most strains of mice, indicating that signals other than those conferred by bcl-2 are required for long-term survival and differentiation of self-reactive B cells in vivo. To further examine the factors that are required for the pathogenesis of autoimmune disease, we have assessed the effect of bcl-2 overexpression on the development of host-versus-graft disease, a self-limited model of systemic autoimmune disease. In this model, injection of spleen cells from (C57BL/6 x BALB/c)F1 hybrid mice into BALB/c newborn parental mice induces immunological tolerance to donor tissues and activation of autoreactive F1 donor B cells through interactions provided by allogeneic host CD4+ T cells. BALB/c newborns injected with spleen cells from (C57BL/6 x BALB/c)F1 mice expressing a bcl-2 transgene in B cells developed high levels of anti-single-stranded DNA and a wide range of pathogenic autoantibodies that were not or barely detectable in mice injected with nontransgenic spleen cells. In mice injected with transgenic B cells, the levels of pathogenic autoantibodies remained high during the course of the study and were associated with long-term persistence of donor B cells, development of a severe autoimmune disease, and accelerated mortality. These results demonstrate that bcl-2 can provide survival signals for the maintenance and differentiation of autoreactive B cells, and suggest that both increased B cell survival and T cell help play critical roles in the development of certain forms of systemic autoimmune disease.

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