scholarly journals TrwC-Mediated Site-Specific Recombination Is Controlled by Host Factors Altering Local DNA Topology

2007 ◽  
Vol 189 (24) ◽  
pp. 9037-9043 ◽  
Author(s):  
Carolina Elvira César ◽  
Matxalen Llosa
Keyword(s):  
Promoter Sequence ◽  
Dna Topology ◽  
Integration Host Factor ◽  
Host Factors ◽  
Elongation Complex ◽  
Site Specific ◽  
Double Stranded Dna ◽  
Transcript Elongation ◽  
A Site

ABSTRACT R388 conjugative relaxase TrwC acts as a site-specific recombinase, promoting recombination between two cognate oriTs on double-stranded DNA substrates. The relaxosome component TrwA is also required for efficient recombination. In this work we present data on the in vivo control of this reaction by host proteins that affect local DNA topology. In the absence of TrwA, binding of integration host factor (IHF) to the oriT keeps the recombination levels low, probably by keeping the relaxosome complex, formed at recombination locus 1, in a “closed” conformation. In an IHF-deficient (IHF−) background, the formation of a transcript elongation complex at this locus still hampers recombination. A mutation abating the promoter sequence at locus 1, or repression of transcription by exposure to rifampin, lifts the inhibition imposed on recombination in an IHF− background. We also observe an increase in conjugation efficiency under these conditions. Relieving the inhibition imposed by these host factors allows efficient levels of recombination between short oriT loci in the absence of TrwA. The presence of TrwA counteracts these inhibitory effects. TrwA would then activate both recombination and conjugation by switching the conformation of the relaxosome to an “open” form that exposes single-stranded DNA at the nic site, promoting the initial TrwC nicking reaction.

scholarly journals Thermo-Sensitive Vesicles in Controlled Drug Delivery for Chemotherapy

Pharmaceutics ◽  
2018 ◽  
Vol 10 (3) ◽  
pp. 150 ◽  
Author(s):  
Elisabetta Mazzotta ◽  
Lorena Tavano ◽  
Rita Muzzalupo
Keyword(s):  
Drug Delivery ◽  
Cancer Treatment ◽  
Controlled Drug Delivery ◽  
Solid Tumours ◽  
Site Specific ◽  
Mild Hyperthermia ◽  
Promising Tool ◽  
A Site

Thermo-sensitive vesicles are a promising tool for triggering the release of drugs to solid tumours when used in combination with mild hyperthermia. Responsivity to temperature makes them intelligent nanodevices able to provide a site-specific chemotherapy. Following a brief introduction concerning hyperthermia and its advantageous combination with vesicular systems, recent investigations on thermo-sensitive vesicles useful for controlled drug delivery in cancer treatment are reported in this review. In particular, the influence of bilayer composition on the in vitro and in vivo behaviour of thermo-sensitive formulations currently under investigation have been extensively explored.

scholarly journals Efficient Integration of Recombinant Adeno-Associated Virus DNA Vectors Requires a p5-rep Sequence in cis

2002 ◽  
Vol 76 (11) ◽  
pp. 5411-5421 ◽  
Author(s):  
Nicola J. Philpott ◽  
Catherine Giraud-Wali ◽  
Carolyn Dupuis ◽  
Janette Gomos ◽  
Henry Hamilton ◽  
...  
Keyword(s):  
First Generation ◽  
Promoter Sequence ◽  
Inverted Terminal Repeat ◽  
Sequence Element ◽  
Adeno Associated Virus ◽  
Enhancer Element ◽  
Site Specific ◽  
Site Specific Integration ◽  
Gene Therapy Vectors ◽  
A Site

ABSTRACT The initial aim of this study was to combine attributes of adeno-associated virus (AAV) and adenovirus (Ad) gene therapy vectors to generate an Ad-AAV hybrid vector allowing efficient site-specific integration with Ad vectors. In executing our experimental strategy, we found that, in addition to the known incompatibility of Rep expression and Ad growth, an equally large obstacle was presented by the inefficiency of the integration event when using traditional recombinant AAV (rAAV) vectors. This study has addressed both of these problems. We have shown that a first-generation Ad can be generated that expresses Rep proteins at levels consistent with those found in wild-type AAV (wtAAV) infections and that Rep-mediated AAV persistence can occur in the presence of first-generation Ad vectors. Our finding that traditional rAAV plasmid vectors lack integration potency compared to wtAAV plasmid constructs (10- to 100-fold differences) was unexpected but led to the discovery of a previously unidentified AAV integration enhancer sequence element which functions in cis to an AAV inverted terminal repeat-flanked target gene. rAAV constructs containing left-end AAV sequence, including the p5-rep promoter sequence, integrate efficiently in a site-specific manner. The identification of this novel AAV integration enhancer element is consistent with previous studies, which have indicated that a high frequency of wtAAV recombinant junction formation occurs in the vicinity of the p5 promoter, and recent studies have demonstrated a role for this region in AAV DNA replication. Understanding the contribution of this element to the mechanism of AAV integration will be critical to the use of AAV vectors for targeted gene transfer applications.

scholarly journals Surface Diversity in Mycoplasma agalactiae Is Driven by Site-Specific DNA Inversions within the vpma Multigene Locus

2002 ◽  
Vol 184 (21) ◽  
pp. 5987-5998 ◽  
Author(s):  
Michelle D. Glew ◽  
Marc Marenda ◽  
Renate Rosengarten ◽  
Christine Citti
Keyword(s):  
Promoter Sequence ◽  
Untranslated Regions ◽  
Dna Rearrangements ◽  
Site Specific ◽  
Reading Frame ◽  
Mycoplasma Agalactiae ◽  
Variable Surface ◽  
A Site ◽  
Active Promoter ◽  
Minimal Region

ABSTRACT The ruminant pathogen Mycoplasma agalactiae possesses a family of abundantly expressed variable surface lipoproteins called Vpmas. Phenotypic switches between Vpma members have previously been correlated with DNA rearrangements within a locus of vpma genes and are proposed to play an important role in disease pathogenesis. In this study, six vpma genes were characterized in the M. agalactiae type strain PG2. All vpma genes clustered within an 8-kb region and shared highly conserved 5′ untranslated regions, lipoprotein signal sequences, and short N-terminal sequences. Analyses of the vpma loci from consecutive clonal isolates showed that vpma DNA rearrangements were site specific and that cleavage and strand exchange occurred within a minimal region of 21 bp located within the 5′ untranslated region of all vpma genes. This process controlled expression of vpma genes by effectively linking the open reading frame (ORF) of a silent gene to a unique active promoter sequence within the locus. An ORF (xer1) immediately adjacent to one end of the vpma locus did not undergo rearrangement and had significant homology to a distinct subset of genes belonging to the λ integrase family of site-specific xer recombinases. It is proposed that xer1 codes for a site-specific recombinase that is not involved in chromosome dimer resolution but rather is responsible for the observed vpma-specific recombination in M. agalactiae.

scholarly journals Dual site-specific chemoenzymatic antibody fragment conjugation using CRISPR-based hybridoma engineering

2020 ◽  
Author(s):  
Camille M. Le Gall ◽  
Johan M.S. van der Schoot ◽  
Iván Ramos-Tomillero ◽  
Melek Parlak Khalily ◽  
Floris J. van Dalen ◽  
...  
Keyword(s):  
Antibody Fragment ◽  
Therapeutic Index ◽  
Stable Cell Line ◽  
Hybridoma Cells ◽  
Target Cells ◽  
Site Specific ◽  
Drug Conjugates ◽  
High Yields ◽  
A Site

I.AbstractFunctionalized antibodies and antibody fragments have found applications in the fields of biomedical imaging, theragnostics, and antibody-drug conjugates (ADC). Antibody functionalization is classically achieved by coupling payloads onto lysine or cysteine residues. However, such stochastic strategies typically lead to heterogenous products, bearing a varying number of payloads. This affects bioconjugate efficacy and stability, as well as its in vivo biodistribution, and therapeutic index, while potentially obstructing the binding sites and leading to off-target toxicity. In addition, therapeutic and theragnostic approaches benefit from the possibility to deliver more than one type of cargo to target cells, further challenging stochastic labelling strategies. Thus, bioconjugation methods to reproducibly obtain defined homogenous conjugates bearing multiple different cargo molecules, without compromising target affinity, are in demand. Here, we describe a straightforward CRISPR/Cas9-based strategy to rapidly engineer hybridoma cells to secrete Fab’ fragments bearing two distinct site-specific labelling motifs, which can be separately modified by two different sortase A mutants. We show that sequential genetic editing of the heavy chain (HC) and light chain (LC) loci enables the generation of a stable cell line that secretes a dual tagged Fab’ molecule (DTFab’), which can be easily isolated in high yields. To demonstrate feasibility, we functionalized the DTFab’ with two distinct cargos in a site-specific manner. This technology platform will be valuable in the development of multimodal imaging agents, theragnostics, and next-generation ADCs.

scholarly journals m-Xylene-Responsive Pu-PnifH Hybrid σ54 Promoters That Overcome Physiological Control in Pseudomonas putida KT2442

2005 ◽  
Vol 187 (1) ◽  
pp. 125-134 ◽  
Author(s):  
Manuel Carmona ◽  
Silvia Fernández ◽  
María J. Rodríguez ◽  
Víctor de Lorenzo
Keyword(s):  
Pseudomonas Putida ◽  
Promoter Sequence ◽  
Growth Conditions ◽  
Integration Host Factor ◽  
Vector System ◽  
Wild Type ◽  
Physiological Control ◽  
Accurate Comparison ◽  
Rnap Binding

ABSTRACT The sequences surrounding the −12/−24 motif of the m-xylene-responsive σ54 promoter Pu of the Pseudomonas putida TOL plasmid pWW0 were replaced by various DNA segments of the same size recruited from PnifH σ54 promoter variants known to have various degrees of efficacy and affinity for σ54-RNA polymerase (RNAP). In order to have an accurate comparison of the output in vivo of each of the hybrids, the resulting promoters were recombined at the same location of the chromosome of P. putida KT2442 with a tailored vector system. The promoters included the upstream activation sequence (UAS) for the cognate regulator of the TOL system (XylR) fused to the −12/−24 region of the wild-type PnifH and its higher σ54-RNAP affinity variants PnifH049 and PnifH319. As a control, the downstream region of the glnAp2 promoter (lacking integration host factor) was fused to the XylR UAS as well. When the induction patterns of the corresponding lacZ fusion strains were compared in vivo, we observed that promoters bearing the RNAP binding site of PnifH049 and PnifH319 were not silenced during exponential growth, as is distinctly the case for the wild-type Pu promoter or for the Pu-PnifH variant. Taken together, our results indicate that the promoter sequence(s) spanning the −12/−24 region of Pu dictates the coupling of promoter output to growth conditions.

scholarly journals Investigations of human myosin VI targeting using optogenetically controlled cargo loading

2017 ◽  
Vol 114 (9) ◽  
pp. E1607-E1616 ◽  
Author(s):  
Alexander R. French ◽  
Tobin R. Sosnick ◽  
Ronald S. Rock
Keyword(s):  
Avena Sativa ◽  
Activation Mechanism ◽  
Myosin Vi ◽  
Site Specific ◽  
Specific Location ◽  
Cargo Protein ◽  
Specific Manner ◽  
A Site

Myosins play countless critical roles in the cell, each requiring it to be activated at a specific location and time. To control myosin VI with this specificity, we created an optogenetic tool for activating myosin VI by fusing the light-sensitive Avena sativa phototropin1 LOV2 domain to a peptide from Dab2 (LOVDab), a myosin VI cargo protein. Our approach harnesses the native targeting and activation mechanism of myosin VI, allowing direct inferences on myosin VI function. LOVDab robustly recruits human full-length myosin VI to various organelles in vivo and hinders peroxisome motion in a light-controllable manner. LOVDab also activates myosin VI in an in vitro gliding filament assay. Our data suggest that protein and lipid cargoes cooperate to activate myosin VI, allowing myosin VI to integrate Ca2+, lipid, and protein cargo signals in the cell to deploy in a site-specific manner.

scholarly journals Flp1 may function in the resolution of recombinant DNA intermediates

2019 ◽  
Vol 15 (3) ◽  
pp. 109
Author(s):  
Phung Thi Thu Huong ◽  
Tran Hong Diem ◽  
Nguyen Luong Hieu Hoa ◽  
Vo Thanh Sang ◽  
Le Van Minh ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Drug Sensitivity ◽  
Recombinant Dna ◽  
Alternative Pathway ◽  
Single Copy ◽  
Site Specific ◽  
Dna Damaging Agents ◽  
2 Micron Plasmid ◽  
A Site

Mus81 is a structure-selective endonuclease which constitutes an alternative pathway in parallel with the helicase-topoisomerase Sgs1-Top3-Rmi1 complex to resolve a number of DNA intermediates during DNA replication, repair, and homologous recombination. Previously, it was shown that the N-terminal region of Mus81 was required for its in vivo function in a redundant manner with Sgs1; sgs1Δmus81Δ100N cells are sensitive to DNA damaging agents. In this study, a single-copy suppressor screening to seek for a factor(s) that could rescue the drug sensitivity of sgs1Δmus81Δ100N cells was performed and revealed that Flp1, a site-specific recombinase 1 encoded on the 2-micron plasmid was a suppressor. This result suggests a function of Flp1 in coordination with Mus81 and Sgs1 to resolve the recombinant DNA intermediates.

scholarly journals Highly Divergent RfaH Orthologs from Pathogenic Proteobacteria Can Substitute for Escherichia coli RfaH both In Vivo and In Vitro

2004 ◽  
Vol 186 (9) ◽  
pp. 2829-2840 ◽  
Author(s):  
Heather D. Carter ◽  
Vladimir Svetlov ◽  
Irina Artsimovitch
Keyword(s):  
Escherichia Coli ◽  
Expression Vectors ◽  
Divergent Evolution ◽  
Transcriptional Enhancer ◽  
Elongation Complex ◽  
E Coli ◽  
Serovar Typhimurium ◽  
Transcript Elongation

ABSTRACT The transcriptional enhancer protein RfaH positively regulates production of virulence factors in Escherichia coli and Salmonella enterica serovar Typhimurium via a cis element, ops. Genes coding for RfaH orthologs were identified in conceptually translated genomes of bacterial pathogens, including Vibrio and Yersinia spp. We cloned the rfaH genes from Vibrio cholerae, Yersinia enterocolitica, S. enterica serovar Typhimurium, and Klebsiella pneumoniae into E. coli expression vectors. Purified RfaH orthologs, including the most divergent one from V. cholerae, were readily recruited to the E. coli transcription elongation complex. Postrecruitment stimulation of transcript elongation appeared to vary with the degree of similarity to E. coli RfaH. V. cholerae RfaH was particularly defective in reducing downstream pausing and termination; this defect was substantially alleviated by an increase in its concentration. When overexpressed episomally, all of the rfaH genes complemented the disruption of the chromosomal copy of the E. coli gene. Thus, despite the apparently accelerated divergent evolution of the RfaH proteins, the mechanism of their action is conserved well enough to make them transcriptionally active in the E. coli system.

scholarly journals Development of an In Vitro Integration Assay for the Bacteroides Conjugative Transposon CTnDOT

2002 ◽  
Vol 184 (17) ◽  
pp. 4829-4837 ◽  
Author(s):  
Qi Cheng ◽  
Neil Wesslund ◽  
Nadja B. Shoemaker ◽  
Abigail A. Salyers ◽  
Jeffrey F. Gardner
Keyword(s):  
Recipient Cell ◽  
Antibiotic Resistance Genes ◽  
Integration Host Factor ◽  
Target Site ◽  
Host Factors ◽  
Integrase Gene ◽  
In Vitro System ◽  
Circular Form

ABSTRACT Integrated self-transmissible elements called conjugative transposons (CTns) are responsible for the transfer of antibiotic resistance genes in many different species of bacteria. One of the best characterized of these newly recognized elements is the Bacteroides CTn, CTnDOT. CTnDOT is thought to have a circular transfer intermediate that transfers to and integrates into the genome of the recipient cell. Previous investigations of the mechanism of CTnDOT integration have been hindered by the lack of an in vitro system for checking this model of integration and determining whether the CTnDOT integrase alone was sufficient to catalyze the integration reaction or whether host factors might be involved. We report here the development of an in vitro system in which a plasmid containing the joined ends of CTnDOT integrates into a plasmid carrying a CTnDOT target site. To develop this in vitro system, a His-tagged version of the integrase gene of CTnDOT was cloned and shown to be active in vivo. The protein produced by this construct was partially purified and then added to a reaction mixture that contained the joined ends of the circular form of CTnDOT and a plasmid carrying one of the CTnDOT target sites. Integration was demonstrated by using a fairly simple mixture of components, but integration was stimulated by a Bacteroides extract or by purified Escherichia coli integration host factor. The results of this study demonstrate both that the circular form of CTnDOT is the form that integrates into the target site and that host factors are involved in the integration process.

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