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Proceedings of the National Academy of... Jun 2000We have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted...
We have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s). In this procedure, recombination requires the phage lambda Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy number plasmid. To demonstrate the utility of this approach, we generated PCR products by using primers with 36- to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. By using the respective PCR products, we made 13 different disruptions of chromosomal genes. Mutants of the arcB, cyaA, lacZYA, ompR-envZ, phnR, pstB, pstCA, pstS, pstSCAB-phoU, recA, and torSTRCAD genes or operons were isolated as antibiotic-resistant colonies after the introduction into bacteria carrying a Red expression plasmid of synthetic (PCR-generated) DNA. The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase which is also easily curable. This procedure should be widely useful, especially in genome analysis of E. coli and other bacteria because the procedure can be done in wild-type cells.
Topics: Chromosomes, Bacterial; DNA Nucleotidyltransferases; Escherichia coli; Integrases; Lac Operon; Mutation; Operon; Plasmids; Polymerase Chain Reaction; Recombinases; Recombination, Genetic
PubMed: 10829079
DOI: 10.1073/pnas.120163297 -
Frontiers in Cellular and Infection... 2022After the outbreak of SARS-CoV-2, nucleic acid testing quickly entered people's lives. In addition to the polymerase chain reaction (PCR) which was commonly used in... (Review)
Review
After the outbreak of SARS-CoV-2, nucleic acid testing quickly entered people's lives. In addition to the polymerase chain reaction (PCR) which was commonly used in nucleic acid testing, isothermal amplification methods were also important nucleic acid testing methods. Among several common isothermal amplification methods like displaced amplification, rolling circle amplification, and so on, recombinase polymerase amplification (RPA) was recently paid more attention to. It had the advantages like a simple operation, fast amplification speed, and reaction at 37-42°C, et al. So it was very suitable for field detection. However, there were still some disadvantages to RPA. Herein, our review mainly summarized the principle, advantages, and disadvantages of RPA. The specific applications of RPA in bacterial detection, fungi detection, virus detection, parasite detection, drug resistance gene detection, genetically modified food detection, and SARS-CoV-2 detection were also described. It was hoped that the latest research progress on RPA could be better delivered to the readers who were interested in RPA.
Topics: Humans; COVID-19; Nucleotidyltransferases; Recombinases; SARS-CoV-2; Sensitivity and Specificity; Nucleic Acid Amplification Techniques
PubMed: 36519130
DOI: 10.3389/fcimb.2022.1019071 -
Nucleic Acids Research Jan 2020Multiplex genetic assays can simultaneously test thousands of genetic variants for a property of interest. However, limitations of existing multiplex assay methods in...
Multiplex genetic assays can simultaneously test thousands of genetic variants for a property of interest. However, limitations of existing multiplex assay methods in cultured mammalian cells hinder the breadth, speed and scale of these experiments. Here, we describe a series of improvements that greatly enhance the capabilities of a Bxb1 recombinase-based landing pad system for conducting different types of multiplex genetic assays in various mammalian cell lines. We incorporate the landing pad into a lentiviral vector, easing the process of generating new landing pad cell lines. We also develop several new landing pad versions, including one where the Bxb1 recombinase is expressed from the landing pad itself, improving recombination efficiency more than 2-fold and permitting rapid prototyping of transgenic constructs. Other versions incorporate positive and negative selection markers that enable drug-based enrichment of recombinant cells, enabling the use of larger libraries and reducing costs. A version with dual convergent promoters allows enrichment of recombinant cells independent of transgene expression, permitting the assessment of libraries of transgenes that perturb cell growth and survival. Lastly, we demonstrate these improvements by assessing the effects of a combinatorial library of oncogenes and tumor suppressors on cell growth. Collectively, these advancements make multiplex genetic assays in diverse cultured cell lines easier, cheaper and more effective, facilitating future studies probing how proteins impact cell function, using transgenic variant libraries tested individually or in combination.
Topics: Animals; Biological Assay; Gene Library; Genetic Vectors; Green Fluorescent Proteins; HEK293 Cells; HT29 Cells; Humans; Lentivirus; Luminescent Proteins; Mice; NIH 3T3 Cells; Oncogenes; Plasmids; Recombinant Proteins; Recombinases; Recombination, Genetic; Transgenes; Tumor Suppressor Proteins; Red Fluorescent Protein
PubMed: 31612958
DOI: 10.1093/nar/gkz910 -
FEMS Microbiology Reviews Aug 2021The integration of mobile genetic elements into their host chromosome influences the immediate fate of cellular organisms and gradually shapes their evolution.... (Review)
Review
The integration of mobile genetic elements into their host chromosome influences the immediate fate of cellular organisms and gradually shapes their evolution. Site-specific recombinases catalyzing this integration have been extensively characterized both in bacteria and eukarya. More recently, a number of reports provided the in-depth characterization of archaeal tyrosine recombinases and highlighted new particular features not observed in the other two domains. In addition to being active in extreme environments, archaeal integrases catalyze reactions beyond site-specific recombination. Some of these integrases can catalyze low-sequence specificity recombination reactions with the same outcome as homologous recombination events generating deep rearrangements of their host genome. A large proportion of archaeal integrases are termed suicidal due to the presence of a specific recombination target within their own gene. The paradoxical maintenance of integrases that disrupt their gene upon integration implies novel mechanisms for their evolution. In this review, we assess the diversity of the archaeal tyrosine recombinases using a phylogenomic analysis based on an exhaustive similarity network. We outline the biochemical, ecological and evolutionary properties of these enzymes in the context of the families we identified and emphasize similarities and differences between archaeal recombinases and their bacterial and eukaryal counterparts.
Topics: Archaea; Eukaryota; Integrases; Recombinases; Tyrosine
PubMed: 33524101
DOI: 10.1093/femsre/fuab004 -
Mammalian Genome : Official Journal of... Mar 2022Recombinase alleles and transgenes can be used to facilitate spatio-temporal specificity of gene disruption or transgene expression. However, the versatility of this in... (Review)
Review
Recombinase alleles and transgenes can be used to facilitate spatio-temporal specificity of gene disruption or transgene expression. However, the versatility of this in vivo recombination system relies on having detailed and accurate characterization of recombinase expression and activity to enable selection of the appropriate allele or transgene. The CrePortal ( http://www.informatics.jax.org/home/recombinase ) leverages the informatics infrastructure of Mouse Genome Informatics to integrate data from the scientific literature, direct data submissions from the scientific community at-large, and from major projects developing new recombinase lines and characterizing recombinase expression and specificity patterns. Searching the CrePortal by recombinase activity or specific recombinase gene driver provides users with a recombinase alleles and transgenes activity tissue summary and matrix comparison of gene expression and recombinase activity with links to generation details, a recombinase activity grid, and associated phenotype annotations. Future improvements will add cell type-based activity annotations. The CrePortal provides a comprehensive presentation of recombinase allele and transgene data to assist researchers in selection of the recombinase allele or transgene based on where and when recombination is desired.
Topics: Alleles; Animals; Integrases; Mice; Mice, Transgenic; Recombinases; Transgenes
PubMed: 34482425
DOI: 10.1007/s00335-021-09909-w -
Microbiology Spectrum Dec 2014In site-specific recombination, two short DNA sequences ('sites') are each cut at specific points in both strands, and the cut ends are rejoined to new partners. The... (Review)
Review
In site-specific recombination, two short DNA sequences ('sites') are each cut at specific points in both strands, and the cut ends are rejoined to new partners. The enzymes that mediate recognition of the sites and the subsequent cutting and rejoining steps are called recombinases. Most recombinases fall into one of two families according to similarities of their protein sequences and mechanisms; these families are known as the tyrosine recombinases and the serine recombinases, the names referring to the conserved amino acid residue that attacks the DNA phosphodiester and becomes covalently linked to a DNA strand end during catalysis. This chapter gives an overview of our current understanding of the serine recombinases, their types, biological roles, structures, catalytic mechanisms, mechanisms of regulation, and applications.
Topics: DNA; Recombinases; Recombination, Genetic; Serine
PubMed: 26104451
DOI: 10.1128/microbiolspec.MDNA3-0046-2014 -
Microbiology Spectrum Dec 2014Covalently closed hairpin ends, also known as hairpin telomeres, provide an unusual solution to the end replication problem. The hairpin telomeres are generated from... (Review)
Review
Covalently closed hairpin ends, also known as hairpin telomeres, provide an unusual solution to the end replication problem. The hairpin telomeres are generated from replication intermediates by a process known as telomere resolution. This is a DNA breakage and reunion reaction promoted by hairpin telomere resolvases (also referred to as protelomerases) found in a limited number of phage and bacteria. The reaction promoted by these enzymes is a chemically isoenergetic two-step transesterification without a requirement for divalent metal ions or high-energy cofactors and uses an active site and mechanism similar to that for type IB topoisomerases and tyrosine recombinases. The small number of unrelated telomere resolvases characterized to date all contain a central, catalytic core domain with the active site, but in addition carry variable C- and N-terminal domains with different functions. Similarities and differences in the structure and function of the telomere resolvases are discussed. Of particular interest are the properties of the Borrelia telomere resolvases, which have been studied most extensively at the biochemical level and appear to play a role in shaping the unusual segmented genomes in these organisms and, perhaps, to play a role in recombinational events.
Topics: Bacteria; Bacteriophages; Catalytic Domain; Genetic Variation; Recombinases; Telomere
PubMed: 26104454
DOI: 10.1128/microbiolspec.MDNA3-0023-2014 -
Microbiology Spectrum Apr 2015Retrotransposons carrying tyrosine recombinases (YR) are widespread in eukaryotes. The first described tyrosine recombinase mobile element, DIRS1, is a retroelement from... (Review)
Review
Retrotransposons carrying tyrosine recombinases (YR) are widespread in eukaryotes. The first described tyrosine recombinase mobile element, DIRS1, is a retroelement from the slime mold Dictyostelium discoideum. The YR elements are bordered by terminal repeats related to their replication via free circular dsDNA intermediates. Site-specific recombination is believed to integrate the circle without creating duplications of the target sites. Recently a large number of YR retrotransposons have been described, including elements from fungi (mucorales and basidiomycetes), plants (green algae) and a wide range of animals including nematodes, insects, sea urchins, fish, amphibia and reptiles. YR retrotransposons can be divided into three major groups: the DIRS elements, PAT-like and the Ngaro elements. The three groups form distinct clades on phylogenetic trees based on alignments of reverse transcriptase/ribonuclease H (RT/RH) and YR sequences, and also having some structural distinctions. A group of eukaryote DNA transposons, cryptons, also carry tyrosine recombinases. These DNA transposons do not encode a reverse transcriptase. They have been detected in several pathogenic fungi and oomycetes. Sequence comparisons suggest that the crypton YRs are related to those of the YR retrotransposons. We suggest that the YR retrotransposons arose from the combination of a crypton-like YR DNA transposon and the RT/RH encoding sequence of a retrotransposon. This acquisition must have occurred at a very early point in the evolution of eukaryotes.
Topics: DNA Replication; Eukaryota; Evolution, Molecular; Genetic Variation; Phylogeny; Recombinases; Recombination, Genetic; Retroelements
PubMed: 26104693
DOI: 10.1128/microbiolspec.MDNA3-0036-2014 -
Biochemical Society Transactions Apr 2010A remarkable feature of the serine resolvases is their regulation: the wild-type enzymes will catalyse intra- but not inter-molecular recombination, can sense the... (Review)
Review
A remarkable feature of the serine resolvases is their regulation: the wild-type enzymes will catalyse intra- but not inter-molecular recombination, can sense the relative orientation of their sites and can exchange strands directionally, despite the fact that there is no net release of chemical bond energy. The key to this regulation is that they are only active within a large intertwined complex called the 'synaptosome'. Because substrate topology greatly facilitates (or, in other cases, inhibits) formation of the synaptosome, it acts as a 'topological filter'. Within the defined topology of the synaptosome, strand exchange releases supercoiling tension, providing an energy source to bias the reaction direction. The regulatory portion of this complex contains additional copies of the recombinase and sometimes other DNA-bending proteins. We are using a combination of X-ray crystallography, biochemistry and genetics to model the full synaptic complex and to understand how the regulatory portion activates the crossover-site-bound recombinases.
Topics: Animals; Bacterial Proteins; DNA; DNA Nucleotidyltransferases; Enzyme Activation; Humans; Models, Biological; Models, Molecular; Nucleic Acid Conformation; Protein Conformation; Recombinases; Recombination, Genetic; Serine
PubMed: 20298188
DOI: 10.1042/BST0380384 -
Plant Biotechnology Journal Feb 2016Efficient methods of stacking genes into plant genomes are needed to expedite transfer of multigenic traits to crop varieties of diverse ecosystems. Over two decades of... (Review)
Review
Efficient methods of stacking genes into plant genomes are needed to expedite transfer of multigenic traits to crop varieties of diverse ecosystems. Over two decades of research has identified several DNA recombinases that carryout efficient cis and trans recombination between the recombination sites artificially introduced into the plant chromosome. The specificity and efficiency of recombinases make them extremely attractive for genome engineering. In plant biotechnology, recombinases have mostly been used for removing selectable marker genes and have rarely been extended to more complex applications. The reversibility of recombination, a property of the tyrosine family of recombinases, does not lend itself to gene stacking approaches that involve rounds of transformation for integrating genes into the engineered sites. However, recent developments in the field of recombinases have overcome these challenges and paved the way for gene stacking. Some of the key advancements include the application of unidirectional recombination systems, modification of recombination sites and transgene site modifications to allow repeated site-specific integrations into the selected site. Gene stacking is relevant to agriculturally important crops, many of which are difficult to transform; therefore, development of high-efficiency gene stacking systems will be important for its application on agronomically important crops, and their elite varieties. Recombinases, by virtue of their specificity and efficiency in plant cells, emerge as powerful tools for a variety of applications including gene stacking.
Topics: Base Sequence; Biotechnology; Gene Expression; Genetic Markers; Recombinases; Recombination, Genetic
PubMed: 26332944
DOI: 10.1111/pbi.12459