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PLoS Genetics Sep 2019In all kingdoms of life, DNA is used to encode hereditary information. Propagation of the genetic material between generations requires timely and accurate duplication... (Review)
Review
In all kingdoms of life, DNA is used to encode hereditary information. Propagation of the genetic material between generations requires timely and accurate duplication of DNA by semiconservative replication prior to cell division to ensure each daughter cell receives the full complement of chromosomes. DNA synthesis of daughter strands starts at discrete sites, termed replication origins, and proceeds in a bidirectional manner until all genomic DNA is replicated. Despite the fundamental nature of these events, organisms have evolved surprisingly divergent strategies that control replication onset. Here, we discuss commonalities and differences in replication origin organization and recognition in the three domains of life.
Topics: Biological Evolution; Cell Division; Chromosomes; DNA Replication; Evolution, Molecular; Replication Origin; Replicon
PubMed: 31513569
DOI: 10.1371/journal.pgen.1008320 -
Science (New York, N.Y.) Nov 2021Molecular virology tools are critical for basic studies of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and for developing new therapeutics....
Molecular virology tools are critical for basic studies of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and for developing new therapeutics. Experimental systems that do not rely on viruses capable of spread are needed for potential use in lower-containment settings. In this work, we use a yeast-based reverse genetics system to develop spike-deleted SARS-CoV-2 self-replicating RNAs. These noninfectious self-replicating RNAs, or replicons, can be trans-complemented with viral glycoproteins to generate replicon delivery particles for single-cycle delivery into a range of cell types. This SARS-CoV-2 replicon system represents a convenient and versatile platform for antiviral drug screening, neutralization assays, host factor validation, and viral variant characterization.
Topics: Animals; Antibodies, Neutralizing; Antibodies, Viral; Antiviral Agents; Cell Line; Humans; Interferons; Microbial Sensitivity Tests; Mutation; Plasmids; RNA, Viral; Replicon; Reverse Genetics; SARS-CoV-2; Saccharomyces cerevisiae; Spike Glycoprotein, Coronavirus; Viral Nonstructural Proteins; Viral Pseudotyping; Virion; Virus Replication
PubMed: 34648371
DOI: 10.1126/science.abj8430 -
Biochemical and Biophysical Research... Dec 2022Nearly 70 years after the proposal of semiconservative replication of generic material by Watson and Crick, we now understand many of the proteins involved in the... (Review)
Review
Nearly 70 years after the proposal of semiconservative replication of generic material by Watson and Crick, we now understand many of the proteins involved in the replication of host chromosomes and how they operate. The initiator and replicator, proposed in the replicon hypothesis, are now well defined in both prokaryotes and eukaryotes. On the other hand, studies in prokaryotes and Archaea indicate alternative modes of initiation, which may not depend on an initiator. Here I summarize recent progress in the field of DNA replication and discuss the evolution of replication systems.
Topics: Replication Origin; DNA Replication; Escherichia coli; DNA-Binding Proteins; Replicon; Bacterial Proteins; Chromosomes, Bacterial; DNA, Bacterial
PubMed: 36344169
DOI: 10.1016/j.bbrc.2022.09.060 -
Cell Stem Cell Aug 2013The generation of human induced pluripotent stem cells (iPSCs) holds great promise for the development of regenerative medicine therapies to treat a wide range of human...
The generation of human induced pluripotent stem cells (iPSCs) holds great promise for the development of regenerative medicine therapies to treat a wide range of human diseases. However, the generation of iPSCs in the absence of integrative DNA vectors remains problematic. Here, we report a simple, highly reproducible RNA-based iPSC generation approach that utilizes a single, synthetic self-replicating VEE-RF RNA replicon that expresses four reprogramming factors (OCT4, KLF4, and SOX2, with c-MYC or GLIS1) at consistent high levels prior to regulated RNA degradation. A single VEE-RF RNA transfection into newborn or adult human fibroblasts resulted in efficient generation of iPSCs with all the hallmarks of stem cells, including cell surface markers, global gene expression profiles, and in vivo pluripotency, to differentiate into all three germ layers. The VEE-RF RNA-based approach has broad applicability for the generation of iPSCs for ultimate use in human stem cell therapies in regenerative medicine.
Topics: Adult; Animals; Cell Differentiation; Cell Line; Cellular Reprogramming; Clone Cells; Fibroblasts; Humans; Induced Pluripotent Stem Cells; Kruppel-Like Factor 4; Male; Mice; Mice, Nude; RNA; Replicon; Transfection
PubMed: 23910086
DOI: 10.1016/j.stem.2013.06.001 -
Microbial Genomics Aug 2018Large-scale bacterial population genetics studies are now routine due to cost-effective Illumina short-read sequencing. However, analysing plasmid content remains...
Large-scale bacterial population genetics studies are now routine due to cost-effective Illumina short-read sequencing. However, analysing plasmid content remains difficult due to incomplete assembly of plasmids. Bacterial isolates can contain any number of plasmids and assembly remains complicated due to the presence of repetitive elements. Numerous tools have been developed to analyse plasmids but the performance and functionality of the tools are variable. The MOB-suite was developed as a set of modular tools for reconstruction and typing of plasmids from draft assembly data to facilitate characterization of plasmids. Using a set of closed genomes with publicly available Illumina data, the MOB-suite identified contigs of plasmid origin with both high sensitivity and specificity (95 and 88 %, respectively). In comparison, plasmidfinder demonstrated high specificity (99 %) but limited sensitivity (50 %). Using the same dataset of 377 known plasmids, MOB-recon accurately reconstructed 207 plasmids so that they were assigned to a single grouping without other plasmid or chromosomal sequences, whereas plasmidSPAdes was only able to accurately reconstruct 102 plasmids. In general, plasmidSPAdes has a tendency to merge different plasmids together, with 208 plasmids undergoing merge events. The MOB-suite reduces the number of errors but produces more hybrid plasmids, with 84 plasmids undergoing both splits and merges. The MOB-suite also provides replicon typing similar to plasmidfinder but with the inclusion of relaxase typing and prediction of conjugation potential. The MOB-suite is written in Python 3 and is available from https://github.com/phac-nml/mob-suite.
Topics: Bacteria; Genome, Bacterial; Plasmids; Replicon; Sequence Analysis, DNA; Software
PubMed: 30052170
DOI: 10.1099/mgen.0.000206 -
Antimicrobial Agents and Chemotherapy Jul 2014In the work presented here, we designed and developed two easy-to-use Web tools for in silico detection and characterization of whole-genome sequence (WGS) and...
In the work presented here, we designed and developed two easy-to-use Web tools for in silico detection and characterization of whole-genome sequence (WGS) and whole-plasmid sequence data from members of the family Enterobacteriaceae. These tools will facilitate bacterial typing based on draft genomes of multidrug-resistant Enterobacteriaceae species by the rapid detection of known plasmid types. Replicon sequences from 559 fully sequenced plasmids associated with the family Enterobacteriaceae in the NCBI nucleotide database were collected to build a consensus database for integration into a Web tool called PlasmidFinder that can be used for replicon sequence analysis of raw, contig group, or completely assembled and closed plasmid sequencing data. The PlasmidFinder database currently consists of 116 replicon sequences that match with at least at 80% nucleotide identity all replicon sequences identified in the 559 fully sequenced plasmids. For plasmid multilocus sequence typing (pMLST) analysis, a database that is updated weekly was generated from www.pubmlst.org and integrated into a Web tool called pMLST. Both databases were evaluated using draft genomes from a collection of Salmonella enterica serovar Typhimurium isolates. PlasmidFinder identified a total of 103 replicons and between zero and five different plasmid replicons within each of 49 S. Typhimurium draft genomes tested. The pMLST Web tool was able to subtype genomic sequencing data of plasmids, revealing both known plasmid sequence types (STs) and new alleles and ST variants. In conclusion, testing of the two Web tools using both fully assembled plasmid sequences and WGS-generated draft genomes showed them to be able to detect a broad variety of plasmids that are often associated with antimicrobial resistance in clinically relevant bacterial pathogens.
Topics: Computer Simulation; Databases, Genetic; Drug Resistance, Bacterial; Enterobacteriaceae; Genome, Bacterial; Internet; Multilocus Sequence Typing; Plasmids; Replicon; Reproducibility of Results
PubMed: 24777092
DOI: 10.1128/AAC.02412-14 -
Microbiology Spectrum Apr 2015Serine resolvases are an interesting group of site-specific recombinases that, in their native contexts, resolve large fused replicons into smaller separated ones. Some... (Review)
Review
Serine resolvases are an interesting group of site-specific recombinases that, in their native contexts, resolve large fused replicons into smaller separated ones. Some resolvases are encoded by replicative transposons and resolve the transposition product, in which the donor and recipient molecules are fused, into separate replicons. Other resolvases are encoded by plasmids and function to resolve plasmid dimers into monomers. Both types are therefore involved in the spread and maintenance of antibiotic-resistance genes. Resolvases and the closely related invertases were the first serine recombinases to be studied in detail, and much of our understanding of the unusual strand exchange mechanism of serine recombinases is owed to those early studies. Resolvases and invertases have also served as paradigms for understanding how DNA topology can be harnessed to regulate enzyme activity. Finally, their relatively modular structure, combined with a wealth of structural and biochemical data, has made them good choices for engineering chimeric recombinases with designer specificity. This chapter focuses on the current understanding of serine resolvases, with a focus on the contributions of structural studies.
Topics: DNA; DNA Transposable Elements; Plasmids; Recombinases; Recombination, Genetic; Replicon; Substrate Specificity
PubMed: 26104713
DOI: 10.1128/microbiolspec.MDNA3-0045-2014 -
Methods in Molecular Biology (Clifton,... 2017Self-replicating RNA derived from the genomes of positive strand RNA viruses represents a powerful tool for both molecular studies on virus biology and approaches to... (Review)
Review
Self-replicating RNA derived from the genomes of positive strand RNA viruses represents a powerful tool for both molecular studies on virus biology and approaches to novel safe and effective vaccines. The following chapter summarizes the principles how such RNAs can be established and used for design of vaccines. Due to the large variety of strategies needed to circumvent specific pitfalls in the design of such constructs the technical details of the experiments are not described here but can be found in the cited literature.
Topics: Animals; Humans; RNA Viruses; RNA, Viral; Replicon; Vaccines
PubMed: 27987141
DOI: 10.1007/978-1-4939-6481-9_2 -
Journal of Medical Virology Jul 2022The ongoing pandemic of coronavirus disease 2019 (COVID-19) has caused severe public health crises and heavy economic losses. Limited knowledge about this deadly virus... (Review)
Review
The ongoing pandemic of coronavirus disease 2019 (COVID-19) has caused severe public health crises and heavy economic losses. Limited knowledge about this deadly virus impairs our capacity to set up a toolkit against it. Thus, more studies on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) biology are urgently needed. Reverse genetics systems, including viral infectious clones and replicons, are powerful platforms for viral research projects, spanning many aspects such as the rescues of wild-type or mutant viral particles, the investigation of viral replication mechanism, the characterization of viral protein functions, and the studies on viral pathogenesis and antiviral drug development. The operations on viral infectious clones are strictly limited in the Biosafety Level 3 (BSL3) facilities, which are insufficient, especially during the pandemic. In contrast, the operation on the noninfectious replicon can be performed in Biosafety Level 2 (BSL2) facilities, which are widely available. After the outbreak of COVID-19, many reverse genetics systems for SARS-CoV-2, including infectious clones and replicons are developed and given plenty of options for researchers to pick up according to the requirement of their research works. In this review, we summarize the available reverse genetics systems for SARS-CoV-2, by highlighting the features of these systems, and provide a quick guide for researchers, especially those without ample experience in operating viral reverse genetics systems.
Topics: COVID-19; Humans; Pandemics; Replicon; Reverse Genetics; SARS-CoV-2
PubMed: 35324008
DOI: 10.1002/jmv.27738 -
EcoSal Plus Jun 2019Plasmids are ubiquitous in the microbial world and have been identified in almost all species of bacteria that have been examined. Their localization inside the... (Review)
Review
Plasmids are ubiquitous in the microbial world and have been identified in almost all species of bacteria that have been examined. Their localization inside the bacterial cell has been examined for about two decades; typically, they are not randomly distributed, and their positioning depends on copy number and their mode of segregation. Low-copy-number plasmids promote their own stable inheritance in their bacterial hosts by encoding active partition systems, which ensure that copies are positioned in both halves of a dividing cell. High-copy plasmids rely on passive diffusion of some copies, but many remain clustered together in the nucleoid-free regions of the cell. Here we review plasmid localization and partition (Par) systems, with particular emphasis on plasmids from and on recent results describing the localization properties and molecular mechanisms of each system. Partition systems also cause plasmid incompatibility such that distinct plasmids (with different replicons) with the same Par system cannot be stably maintained in the same cells. We discuss how partition-mediated incompatibility is a consequence of the partition mechanism.
Topics: Bacterial Proteins; Enterobacteriaceae; Plasmids; Replicon
PubMed: 31187729
DOI: 10.1128/ecosalplus.ESP-0003-2019