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Research in Microbiology 2000Both Neisseria meningitidis and Streptococcus pneumoniae are naturally transformable species and are known to be freely recombining in the wild. Large multilocus... (Comparative Study)
Comparative Study Review
Both Neisseria meningitidis and Streptococcus pneumoniae are naturally transformable species and are known to be freely recombining in the wild. Large multilocus sequence typing (MLST) datasets have been generated for these species. Here we outline an approach which exploits these data sets in order to quantify the extent of recombination, thus enabling meaningful comparisons between the two species. Two parameters are estimated; the rate at which recombination changes alleles, compared to point mutation, and the rate at which recombination changes individual nucleotide sites, compared to point mutation. Estimates for the former parameter are 4:1 in the meningococcus (i.e. alleles are changed four-fold more frequently by recombination than by mutation), and 10:1 in the pneumococcus. However, estimates for the latter parameter are at least 80:1 in the meningococcus (i.e. an individual nucleotide site is at least 80-fold more likely to change by recombination than by mutation) and 50:1 in the pneumococcus. These data imply that recombination events, compared to mutational events, may be more common in the pneumococcus than in the meningococcus. However, because it is a more diverse species, each recombinational exchange in the meningococcus results in more nucleotide changes on average.
Topics: Genetic Variation; Models, Genetic; Neisseria meningitidis; Point Mutation; Recombination, Genetic; Streptococcus pneumoniae
PubMed: 10961460
DOI: 10.1016/s0923-2508(00)00168-6 -
Molecular Cell Dec 2001The recombination mechanisms that deal with double-strand breaks in organisms as diverse as phage, bacteria, yeast, and humans are remarkably conserved. We discuss... (Review)
Review
The recombination mechanisms that deal with double-strand breaks in organisms as diverse as phage, bacteria, yeast, and humans are remarkably conserved. We discuss conservation in the biochemical pathways required to recombine DNA ends and in the structure of the DNA products. In addition, we highlight that two fundamentally distinct broken DNA substrates exist and describe how they are repaired differently by recombination. Finally, we discuss the need to coordinate recombinational repair with cell division through DNA damage response pathways.
Topics: Animals; Bacteriophage T4; DNA; DNA Damage; DNA Repair; DNA Replication; Escherichia coli; Eukaryotic Cells; Evolution, Molecular; Humans; Models, Genetic; Recombination, Genetic; Sequence Homology, Nucleic Acid
PubMed: 11779493
DOI: 10.1016/s1097-2765(01)00419-1 -
PloS One 2011Genetic manipulation of poxvirus genomes through attenuation, or insertion of therapeutic genes has led to a number of vector candidates for the treatment of a variety...
BACKGROUND
Genetic manipulation of poxvirus genomes through attenuation, or insertion of therapeutic genes has led to a number of vector candidates for the treatment of a variety of human diseases. The development of recombinant poxviruses often involves the genomic insertion of a selectable marker for purification and selection purposes. The use of marker genes however inevitably results in a vector that contains unwanted genetic information of no therapeutic value.
METHODOLOGY/PRINCIPAL FINDINGS
Here we describe an improved strategy that allows for the creation of marker-free recombinant poxviruses of any species. The Selectable and Excisable Marker (SEM) system incorporates a unique fusion marker gene for the efficient selection of poxvirus recombinants and the Cre/loxP system to facilitate the subsequent removal of the marker. We have defined and characterized this new methodological tool by insertion of a foreign gene into vaccinia virus, with the subsequent removal of the selectable marker. We then analyzed the importance of loxP orientation during Cre recombination, and show that the SEM system can be used to introduce site-specific deletions or inversions into the viral genome. Finally, we demonstrate that the SEM strategy is amenable to other poxviruses, as demonstrated here with the creation of an ectromelia virus recombinant lacking the EVM002 gene.
CONCLUSION/SIGNIFICANCE
The system described here thus provides a faster, simpler and more efficient means to create clinic-ready recombinant poxviruses for therapeutic gene therapy applications.
Topics: Animals; Cattle; Cell Line; Cell Line, Tumor; Chlorocebus aethiops; Genetic Markers; Genetic Vectors; Humans; Poxviridae; Recombination, Genetic
PubMed: 21931792
DOI: 10.1371/journal.pone.0024643 -
Journal of Virology Jul 2015Varicella-zoster virus (VZV) is a human herpesvirus, which during primary infection typically causes varicella (chicken pox) and establishes lifelong latency in sensory...
UNLABELLED
Varicella-zoster virus (VZV) is a human herpesvirus, which during primary infection typically causes varicella (chicken pox) and establishes lifelong latency in sensory and autonomic ganglia. Later in life, the virus may reactivate to cause herpes zoster (HZ; also known as shingles). To prevent these diseases, a live-attenuated heterogeneous vaccine preparation, vOka, is used routinely in many countries worldwide. Recent studies of another alphaherpes virus, infectious laryngotracheitis virus, demonstrate that live-attenuated vaccine strains can recombine in vivo, creating virulent progeny. These findings raised concerns about using attenuated herpesvirus vaccines under conditions that favor recombination. To investigate whether VZV may undergo recombination, which is a prerequisite for VZV vaccination to create such conditions, we here analyzed 115 complete VZV genomes. Our results demonstrate that recombination occurs frequently for VZV. It thus seems that VZV is fully capable of recombination if given the opportunity, which may have important implications for continued VZV vaccination. Although no interclade vaccine wild-type recombinant strains were found, intraclade recombinants were frequently detected in clade 2, which harbors the vaccine strains, suggesting that the vaccine strains have already been involved in recombination events, either in vivo or in vitro during passages in cell culture. Finally, previous partial and complete genomic studies have described strains that do not cluster phylogenetically to any of the five established clades. The additional VZV strains sequenced here, in combination with those previously published, have enabled us to formally define a novel sixth VZV clade.
IMPORTANCE
Although genetic recombination has been demonstrated to frequently occur for other human alphaherpesviruses, herpes simplex viruses 1 and 2, only a few ancient and isolated recent recombination events have hitherto been demonstrated for VZV. In the present study, we demonstrate that VZV also frequently undergoes genetic recombination, including strains belonging to the clade containing the vOKA strain.
Topics: Adult; Child; Child, Preschool; Cluster Analysis; DNA, Viral; Genetic Variation; Genome, Viral; Herpesvirus 3, Human; Humans; Molecular Sequence Data; Phylogeny; Recombination, Genetic; Sequence Analysis, DNA; Sequence Homology
PubMed: 25926648
DOI: 10.1128/JVI.00437-15 -
PloS One 2013Tomato yellow leaf curl virus (TYLCV) is a highly damaging begomovirus native to the Middle East. TYLCV has recently spread worldwide, recombining with other...
Tomato yellow leaf curl virus (TYLCV) is a highly damaging begomovirus native to the Middle East. TYLCV has recently spread worldwide, recombining with other begomoviruses. Recent analysis of mixed infections between TYLCV and Tomato leaf curl Comoros begomovirus (ToLCKMV) has shown that, although natural selection preserves certain co-evolved intra-genomic interactions, numerous and diverse recombinants are produced at 120 days post-inoculation (dpi), and recombinant populations from different tomato plants are very divergent. Here, we investigate the population dynamics that lead to such patterns in tomato plants co-infected with TYLCV and ToLCKMV either by agro-inoculation or using the natural whitefly vector Bemisia tabaci. We monitored the frequency of parental and recombinant genotypes independently in 35 plants between 18 and 330 dpi and identified 177 recombinants isolated at different times. Recombinants were detected from 18 dpi and their frequency increased over time to reach about 50% at 150 dpi regardless of the inoculation method. The distribution of breakpoints detected on 96 fully sequenced recombinants was consistent with a continuous generation of new recombinants as well as random and deterministic effects in their maintenance. A severe population bottleneck of around 10 genomes was estimated during early systemic infection-a phenomenon that could account partially for the heterogeneity in recombinant patterns observed among plants. The detection of the same recombinant genome in six of the thirteen plants analysed beyond 30 dpi supported the influence of selection on observed recombination patterns. Moreover, a highly virulent recombinant genotype dominating virus populations within one plant has, apparently, the potential to be maintained in the natural population according to its infectivity, within-host accumulation, and transmission efficiency - all of which were similar or intermediate to those of the parent genotypes. Our results anticipate the outcomes of natural encounters between TYLCV and ToLCKMV.
Topics: Animals; Begomovirus; Evolution, Molecular; Genome, Viral; Genomics; Genotype; Hemiptera; Solanum lycopersicum; Plant Diseases; Plant Leaves; Recombination, Genetic; Virulence
PubMed: 23472190
DOI: 10.1371/journal.pone.0058375 -
Methods in Molecular Biology (Clifton,... 2018Here, we describe a methodology that allows the insertion of site-specific DNA lesions into genomes in living cells. The technique involves the integration of a plasmid...
Here, we describe a methodology that allows the insertion of site-specific DNA lesions into genomes in living cells. The technique involves the integration of a plasmid containing a site-specific lesion engineered in vitro into a precise location in the genome via the site-specific recombination reaction from phage lambda. The notion of DNA lesion is not restricted to chemically modified nucleotides but also refers to unusual DNA structures. This method will be instrumental to study qualitatively and quantitatively the genetic consequences of site-specific lesions in vivo; moreover, it does also allow analyzing the molecular structure of stalled replication forks at well-defined locations.
Topics: DNA Damage; DNA Replication; Escherichia coli; Genes, Reporter; Genome; Plasmids; Recombination, Genetic; Recombinational DNA Repair
PubMed: 29043620
DOI: 10.1007/978-1-4939-7306-4_9 -
Applied and Environmental Microbiology Jan 2021Genetic recombination plays a pivotal role in the appearance of human norovirus recombinants that cause global epidemics. However, the factors responsible for the...
Genetic recombination plays a pivotal role in the appearance of human norovirus recombinants that cause global epidemics. However, the factors responsible for the appearance of these recombinants remains largely unknown. In this study, we revealed a selective pressure that restricts parental combinations leading to the emergence of norovirus recombinants. To investigate traces of emerging novel recombinants and their parents in the human population, we isolated mass nucleotide sequence clones of human norovirus genogroups I and II in sewage-affected waters over a 4-year sampling period. Fourteen different phylogenetic combinations of recombinants and their parents were defined from the dozens of phylogenetic lineages circulating in the human population. To evaluate the probability of these combinations, parental lineages of each recombinant were categorized into two groups as HP (relatively higher-competitiveness parents) and LP (relatively lower-competitiveness parents), according to their relative detection frequency. Strong categorization of HP and LP was confirmed by tests with modified data and additional variables. An algorithm that was developed in this study to visualize the chance of mixed infection between parents revealed that HP lineages have a higher chance of mixed infection than LP lineages in the human population. Three parental pairing types in recombinants were defined: HP-HP, HP-LP, and LP-LP. Among these, most recombinants were identified as HP-LP, despite the prediction of dominant emergence of HP-HP-type recombinants. These results suggest that nature favors recombinants of human norovirus that originate from parental pairing of heterogeneous competitiveness. Novel recombinants, generated from inter- and intraspecies recombination of norovirus lineages, often emerge and pose a threat to public health. However, the factors determining emergence of these particular recombinants from all possible combinations of parental lineages remain largely unknown. Therefore, current investigations on these recombinants are inevitably limited to postepidemic analyses, which merely identify genetic or phenotypic changes in the newly emerged recombinants compared to their parents. Here, we provide a new theoretical concept that emergence of novel recombinants could be explained by a combination of parental noroviruses thriving in the human population and those circulating at lower levels. This study could provide an additional and important rationale for the proactive environmental monitoring of potential future epidemics due to viral recombinants.
Topics: Base Sequence; Genotype; Humans; Norovirus; Phylogeny; Recombination, Genetic; Republic of Korea; Sewage
PubMed: 33187997
DOI: 10.1128/AEM.02015-20 -
Current Issues in Molecular Biology Jul 2004Recombination is a ubiquitous genetic process which results in the exchange of DNA between two substrates. Homologous recombination occurs between DNA species with... (Review)
Review
Recombination is a ubiquitous genetic process which results in the exchange of DNA between two substrates. Homologous recombination occurs between DNA species with identical sequence whereas illegitimate recombination can occur between DNA with very little or no homology. Site-specific recombination is often used by temperate phages to stably integrate into bacterial chromosomes. Characterisation of the mechanisms of recombination in mycobacteria has mainly focussed on RecA-dependent homologous recombination and phage-directed site-specific recombination. In contrast the high frequency of illegitimate recombination in slow-growing mycobacteria has not been explained. The role of DNA repair in dormancy and infection have not yet been fully established, but early work suggests that RecA-mediated pathways are not required for virulence. All three recombination mechanisms have been utilised in developing genetic techniques for the analysis of the biology and pathogenesis of mycobacteria. A recently developed method for studying essential genes will generate further insights into the biology of these important organisms.
Topics: DNA, Recombinant; Genetic Markers; Genetic Vectors; Mycobacteriaceae; Recombination, Genetic; Transduction, Genetic
PubMed: 15119825
DOI: No ID Found -
Plant Physiology Dec 2007
Review
Topics: Cloning, Molecular; Genetic Engineering; Genetic Vectors; Genome, Plant; Plants; Recombination, Genetic; Software
PubMed: 18056864
DOI: 10.1104/pp.107.106989 -
Current Protocols Feb 2023The bacterial chromosome and bacterial plasmids can be engineered in vivo by homologous recombination using either PCR products or synthetic double-stranded DNA (dsDNA)...
The bacterial chromosome and bacterial plasmids can be engineered in vivo by homologous recombination using either PCR products or synthetic double-stranded DNA (dsDNA) or single-stranded DNA as substrates. Multiple linear dsDNA molecules can be assembled into an intact plasmid. The technology of recombineering is possible because bacteriophage-encoded recombination proteins efficiently recombine sequences with homologies as short as 35 to 50 bases. Recombineering allows DNA sequences to be inserted or deleted without regard to the location of restriction sites and can also be used in combination with CRISPR/Cas targeting systems. © 2023 Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA. Basic Protocol: Making electrocompetent cells and transforming with linear DNA Support Protocol 1: Selection/counter-selections for genome engineering Support Protocol 2: Creating and screening for oligo recombinants by PCR Support Protocol 3: Other methods of screening for unselected recombinants Support Protocol 4: Curing recombineering plasmids containing a temperature-sensitive replication function Support Protocol 5: Removal of the prophage by recombineering Alternate Protocol 1: Using CRISPR/Cas9 as a counter-selection following recombineering Alternate Protocol 2: Assembly of linear dsDNA fragments into functional plasmids Alternate Protocol 3: Retrieval of alleles onto a plasmid by gap repair Alternate Protocol 4: Modifying multicopy plasmids with recombineering Support Protocol 6: Screening for unselected plasmid recombinants Alternate Protocol 5: Recombineering with an intact λ prophage Alternate Protocol 6: Targeting an infecting λ phage with the defective prophage strains.
Topics: Humans; Escherichia coli; Homologous Recombination; Genetic Engineering; Plasmids; Polymerase Chain Reaction
PubMed: 36779782
DOI: 10.1002/cpz1.656