scholarly journals In vitro amplification of whole large plasmids via transposon-mediated oriC insertion

BioTechniques ◽  
2021 ◽  
Author(s):  
Seia Nara ◽  
Masayuki Su'etsugu
Keyword(s):  
Dna Amplification ◽  
Initiation Site ◽  
Replication Initiation ◽  
Replication Cycle ◽  
Sequence Information ◽  
Circular Dna ◽  
Circular Dnas ◽  
Large Plasmids ◽  
Dna Template

DNA amplification is a fundamental technique in molecular biology. The replication cycle reaction is a new method for amplification of large circular DNA having oriC sequences, which is a replication initiation site of the Escherichia coli chromosome. We here developed a replication cycle reaction-based method useful for amplification of various circular DNAs lacking oriC, even in the absence of any sequence information, via transposon-mediated oriC insertion to the circular DNA template. A 15-kb non- oriC plasmid was amplified from a very small amount of starting DNA (50 fg, 1 fM). The method was also applicable to GC-rich plasmid (69%) or large F-plasmid (230 kb). This method thus provides a powerful tool to amplify various environmental circular DNAs.

scholarly journals Inhibitory Effect of Adefovir on Viral DNA Synthesis and Covalently Closed Circular DNA Formation in Duck Hepatitis B Virus-Infected Hepatocytes In Vivo and In Vitro

2002 ◽  
Vol 46 (2) ◽  
pp. 425-433 ◽  
Author(s):  
Julien Delmas ◽  
Olivier Schorr ◽  
Catherine Jamard ◽  
Craig Gibbs ◽  
Christian Trépo ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Dna Synthesis ◽  
Dna Amplification ◽  
Viral Dna ◽  
Circular Dna ◽  
Covalently Closed Circular Dna ◽  
B Virus

ABSTRACT The elimination of viral covalently closed circular DNA (CCC DNA) from the nucleus of infected hepatocytes is an obstacle to achieving sustained viral clearance during antiviral therapy of chronic hepatitis B virus (HBV) infection. The aim of our study was to determine whether treatment with adefovir, a new acyclic nucleoside phosphonate, the prodrug of which, adefovir dipivoxil, is in clinical evaluation, is able to suppress viral CCC DNA both in vitro and in vivo using the duck HBV (DHBV) model. First, the effect of adefovir on viral CCC DNA synthesis was examined with primary cultures of DHBV-infected fetal hepatocytes. Adefovir was administered for six consecutive days starting one day before or four days after DHBV inoculation. Dose-dependent inhibition of both virion release in culture supernatants and synthesis of intracellular viral DNA was observed. Although CCC DNA amplification was inhibited by adefovir, CCC DNA was not eliminated by antiviral treatment and the de novo formation of CCC DNA was not prevented by pretreatment of the cells. Next, preventive treatment of experimentally infected ducklings with lamivudine or adefovir revealed that both efficiently suppressed viremia and intrahepatic DNA. However, persistence of viral DNA even when detectable only by PCR was associated with a recurrence of viral replication following drug withdrawal. Taken together, our results demonstrate that adefovir is a potent inhibitor of DHBV replication that inhibits CCC DNA amplification but does not effectively prevent the formation of CCC DNA from incoming viral genomes.

scholarly journals ModA and ModB, Two ADP-Ribosyltransferases Encoded by Bacteriophage T4: Catalytic Properties and Mutation Analysis

2004 ◽  
Vol 186 (21) ◽  
pp. 7262-7272 ◽  
Author(s):  
Bernd Tiemann ◽  
Reinhard Depping ◽  
Egle Gineikiene ◽  
Laura Kaliniene ◽  
Rimas Nivinskas ◽  
...  
Keyword(s):  
Bacteriophage T4 ◽  
In Vitro Transcription ◽  
Reaction Scheme ◽  
Replication Cycle ◽  
Site Directed Mutagenesis ◽  
Host Proteins ◽  
Host Interactions ◽  
Late Transcription ◽  
Dna Template

ABSTRACT Bacteriophage T4 encodes three ADP-ribosyltransferases, Alt, ModA, and ModB. These enzymes participate in the regulation of the T4 replication cycle by ADP-ribosylating a defined set of host proteins. In order to obtain a better understanding of the phage-host interactions and their consequences for regulating the T4 replication cycle, we studied cloning, overexpression, and characterization of purified ModA and ModB enzymes. Site-directed mutagenesis confirmed that amino acids, as deduced from secondary structure alignments, are indeed decisive for the activity of the enzymes, implying that the transfer reaction follows the Sn1-type reaction scheme proposed for this class of enzymes. In vitro transcription assays performed with Alt- and ModA-modified RNA polymerases demonstrated that the Alt-ribosylated polymerase enhances transcription from T4 early promoters on a T4 DNA template, whereas the transcriptional activity of ModA-modified polymerase, without the participation of T4-encoded auxiliary proteins for middle mode or late transcription, is reduced. The results presented here support the conclusion that ADP-ribosylation of RNA polymerase and of other host proteins allows initial phage-directed mRNA synthesis reactions to escape from host control. In contrast, subsequent modification of the other cellular target proteins limits transcription from phage early genes and participates in redirecting transcription to phage middle and late genes.

OriCiro® Cell-Free Cloning System/PASS V4.1.2 v1

2021 ◽  
Author(s):  
shigemasa.s not provided
Keyword(s):  
Chromosome Replication ◽  
Replication Cycle ◽  
Restriction Enzyme Digestion ◽  
Dna Fragments ◽  
Circular Dna ◽  
E Coli ◽  
Plasmid Construction ◽  
Cloning System ◽  
Maximum Amplification

OriCiro® Cell-Free Cloning System is a rapid and powerful tool replacing cumbersome DNA cloning (plasmid construction) process relying on E. coli. The system consists of two kits. OriCiro Assembly Kit allows seamless assembly of multiple overlapping DNA fragments. The assembly product can be added directly to OriCiro Amp Kit to get selective amplification of your target circular DNA (Figure 1). The amplified product is supercoiled DNA topologically identical to plasmid DNA isolated from E. coli. 1. OriCiro Assembly Kit: Multiple DNA fragments are assembled seamlessly at 42 ̊C for 30 minutes via ~40 bp overlapping ends (Figure 2). DNA fragments generated by PCR or restriction enzyme digestion are available. Our unique enzyme-based annealing mechanism allows powerful assembly up to 50 fragments simultaneously. 2. OriCiro Amp Kit: The reaction consists of 26 purified enzymes involved in chromosome replication of E. coli. The chromosome replication cycle repeats autonomously at around 30 ̊C, enabling exponential amplification of circular DNA having oriC with extremely high fidelity (10-8 error/base/cycle) (Figure 3). The kit yields up to 1 μg circular DNA per 10 μL reaction at 33 ̊C for 6 hr. The maximum amplification size is 50 kb in the current version of the kit. n.b. OriCiro Amp NEEDS oriC Cassette (0.4 kb) which can be inserted into circular DNA using OriCiro assembly kit. References: 1. T. Mukai, T. Yoneji, K. Yamada, H. Fujita, S. Nara, M. Su'etsugu, Overcoming the Challenges of Megabase-Sized Plasmid Construction in Escherichia coli, ACS Synthetic Biology, 2020, 9 (6), 1315- 1327 2. T. Hasebe, K. Narita, S. Hidaka, M. Su'etsugu, Efficient Arrangement of the Replication Fork Trap for In Vitro Propagation of Monomeric Circular DNA in the Chromosome-Replication Cycle Reaction. Life, 2018, 8 (43) 3. M. Su’etsugu, H. Takada, T. Katayama, H. Tsujimoto, Exponential propagation of large circular DNA by reconstitution of a chromosome-replication cycle, Nucleic Acids Research, 2017, 45 (20), 11525– 11534

scholarly journals Metabolic cofactors NADH and FAD act as non-canonical initiating substrates for a primase and affect replication primer processing in vitro

2020 ◽  
Author(s):  
Christina Julius ◽  
Paula S Salgado ◽  
Yulia Yuzenkova
Keyword(s):  
De Novo ◽  
Response Regulator ◽  
Replication Initiation ◽  
Reduced Form ◽  
Dna Polymerase I ◽  
Base Pairs ◽  
Okazaki Fragments ◽  
Polymerase I ◽  
Dna Template

Abstract To initiate replication on a double-stranded DNA de novo, all organisms require primase, an RNA polymerase making short RNA primers which are then extended by DNA polymerases. Here, we show that primase can use metabolic cofactors as initiating substrates, instead of its canonical substrate ATP. DnaG primase of Escherichia coli initiates synthesis of RNA with NADH (the reduced form of nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide) in vitro. These cofactors consist of an ADP core covalently bound to extra moieties. The ADP component of these metabolites base-pairs with the DNA template and provides a 3′-OH group for RNA extension. The additional cofactors moieties apparently contact the ‘basic ridge’ domain of DnaG, but not the DNA template base at the –1 position. ppGpp, the starvation response regulator, strongly inhibits the initiation with cofactors, hypothetically due to competition for overlapping binding sites. Efficient RNA primer processing is a prerequisite for Okazaki fragments maturation, and we find that the efficiency of primer processing by DNA polymerase I in vitro is specifically affected by the cofactors on its 5′-end. Together these results indicate that utilization of cofactors as substrates by primase may influence regulation of replication initiation and Okazaki fragments processing.

scholarly journals Efficient Arrangement of the Replication Fork Trap for In Vitro Propagation of Monomeric Circular DNA in the Chromosome-Replication Cycle Reaction

Life ◽  
2018 ◽  
Vol 8 (4) ◽  
pp. 43 ◽  
Author(s):  
Tomonori Hasebe ◽  
Kouhei Narita ◽  
Shiomi Hidaka ◽  
Masayuki Su’etsugu
Keyword(s):  
Genetic Information ◽  
Replication Fork ◽  
In Vitro Propagation ◽  
Detrimental Effect ◽  
Living Cells ◽  
Replication Cycle ◽  
Replication Forks ◽  
Circular Dna ◽  
Replication Fork Progression

Propagation of genetic information is a fundamental prerequisite for living cells. We recently developed the replication cycle reaction (RCR), an in vitro reaction for circular DNA propagation, by reconstitution of the replication cycle of the Escherichia coli chromosome. In RCR, two replication forks proceed bidirectionally from the replication origin, oriC, and meet at a region opposite oriC, yielding two copies of circular DNA. Although RCR essentially propagates supercoiled monomers, concatemer byproducts are also produced due to inefficient termination of the replication fork progression. Here, we examined the effect of the Tus-ter replication fork trap in RCR. Unexpectedly, when the fork traps were placed opposite oriC, mimicking their arrangement on the chromosome, the propagation of circular DNA was inhibited. On the other hand, fork traps flanking oriC allowed efficient propagation of circular DNA and repressed concatemer production. These findings suggest that collision of the two convergence forks through the fork trap is detrimental to repetition of the replication cycle. We further demonstrate that this detrimental effect was rescued by the UvrD helicase. These results provide insights into the way in which circular DNA monomers replicate repetitively without generating concatemers.

scholarly journals CirBiTree: Citrullination Site Inference Based on a Fuzzy Neural Network and Flexible Neural Tree

2020 ◽  
Vol 2020 ◽  
pp. 1-8
Author(s):  
Chuandong Song ◽  
Haifeng Wang
Keyword(s):  
Neural Network ◽  
Fuzzy Neural Network ◽  
Classification Model ◽  
Peptide Sequence ◽  
Sequence Information ◽  
Post Translational Modification ◽  
Fuzzy Neural ◽  
Human Complex ◽  
Better Than

Emerging evidence demonstrates that post-translational modification plays an important role in several human complex diseases. Nevertheless, considering the inherent high cost and time consumption of classical and typical in vitro experiments, an increasing attention has been paid to the development of efficient and available computational tools to identify the potential modification sites in the level of protein. In this work, we propose a machine learning-based model called CirBiTree for identification the potential citrullination sites. More specifically, we initially utilize the biprofile Bayesian to extract peptide sequence information. Then, a flexible neural tree and fuzzy neural network are employed as the classification model. Finally, the most available length of identified peptides has been selected in this model. To evaluate the performance of the proposed methods, some state-of-the-art methods have been employed for comparison. The experimental results demonstrate that the proposed method is better than other methods. CirBiTree can achieve 83.07% in sn%, 80.50% in sp, 0.8201 in F1, and 0.6359 in MCC, respectively.

scholarly journals Putative Cooperative ATP–DnaA Binding to Double-Stranded DnaA Box and Single-Stranded DnaA-Trio Motif upon Helicobacter pylori Replication Initiation Complex Assembly

2021 ◽  
Vol 22 (12) ◽  
pp. 6643
Author(s):  
Pawel Jaworski ◽  
Dorota Zyla-Uklejewicz ◽  
Malgorzata Nowaczyk-Cieszewska ◽  
Rafal Donczew ◽  
Thorsten Mielke ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Replication Initiation ◽  
Dna Unwinding ◽  
Rich Region ◽  
Initiator Protein ◽  
New Class ◽  
H Pylori ◽  
General Architecture ◽  
Dnaa Box

oriC is a region of the bacterial chromosome at which the initiator protein DnaA interacts with specific sequences, leading to DNA unwinding and the initiation of chromosome replication. The general architecture of oriCs is universal; however, the structure of oriC and the mode of orisome assembly differ in distantly related bacteria. In this work, we characterized oriC of Helicobacter pylori, which consists of two DnaA box clusters and a DNA unwinding element (DUE); the latter can be subdivided into a GC-rich region, a DnaA-trio and an AT-rich region. We show that the DnaA-trio submodule is crucial for DNA unwinding, possibly because it enables proper DnaA oligomerization on ssDNA. However, we also observed the reverse effect: DNA unwinding, enabling subsequent DnaA–ssDNA oligomer formation—stabilized DnaA binding to box ts1. This suggests the interplay between DnaA binding to ssDNA and dsDNA upon DNA unwinding. Further investigation of the ts1 DnaA box revealed that this box, together with the newly identified c-ATP DnaA box in oriC1, constitute a new class of ATP–DnaA boxes. Indeed, in vitro ATP–DnaA unwinds H. pylori oriC more efficiently than ADP–DnaA. Our results expand the understanding of H. pylori orisome formation, indicating another regulatory pathway of H. pylori orisome assembly.

scholarly journals Revisiting Ehrlichia ruminantium Replication Cycle Using Proteomics: The Host and the Bacterium Perspectives

2021 ◽  
Vol 9 (6) ◽  
pp. 1144
Author(s):  
Isabel Marcelino ◽  
Philippe Holzmuller ◽  
Ana Coelho ◽  
Gabriel Mazzucchelli ◽  
Bernard Fernandez ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Host Cell ◽  
Cell Metabolism ◽  
Replication Cycle ◽  
Host Cells ◽  
Ehrlichia Ruminantium ◽  
Host Interaction ◽  
Infected Cells ◽  
Related Proteins

The Rickettsiales Ehrlichia ruminantium, the causal agent of the fatal tick-borne disease Heartwater, induces severe damage to the vascular endothelium in ruminants. Nevertheless, E. ruminantium-induced pathobiology remains largely unknown. Our work paves the way for understanding this phenomenon by using quantitative proteomic analyses (2D-DIGE-MS/MS, 1DE-nanoLC-MS/MS and biotin-nanoUPLC-MS/MS) of host bovine aorta endothelial cells (BAE) during the in vitro bacterium intracellular replication cycle. We detect 265 bacterial proteins (including virulence factors), at all time-points of the E. ruminantium replication cycle, highlighting a dynamic bacterium–host interaction. We show that E. ruminantium infection modulates the expression of 433 host proteins: 98 being over-expressed, 161 under-expressed, 140 detected only in infected BAE cells and 34 exclusively detected in non-infected cells. Cystoscape integrated data analysis shows that these proteins lead to major changes in host cell immune responses, host cell metabolism and vesicle trafficking, with a clear involvement of inflammation-related proteins in this process. Our findings led to the first model of E. ruminantium infection in host cells in vitro, and we highlight potential biomarkers of E. ruminantium infection in endothelial cells (such as ROCK1, TMEM16K, Albumin and PTPN1), which may be important to further combat Heartwater, namely by developing non-antibiotic-based strategies.

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