The dif/Xer Recombination Systems in Proteobacteria

In E. coli, 10 to 15% of growing bacteria produce dimeric chromosomes during DNA replication. These dimers are resolved by XerC and XerD, two tyrosine recombinases that target the 28-nucleotide motif (dif) associated with the chromosome's replication terminus. In streptococci and lactococci, an alternative system is composed of a unique, Xer-like recombinase (XerS) genetically linked to a dif-like motif (difSL) located at the replication terminus. Preliminary observations have suggested that the dif/Xer system is commonly found in bacteria with circular chromosomes but that assumption has not been confirmed in an exhaustive analysis. The aim of the present study was to extensively characterize the dif/Xer system in the proteobacteria, since this taxon accounts for the majority of genomes sequenced to date. To that end, we analyzed 234 chromosomes from 156 proteobacterial species and showed that most species (87.8%) harbor XerC and XerD-like recombinases and a dif-related sequence which (i) is located in non-coding sequences, (ii) is close to the replication terminus (as defined by the cumulative GC skew) (iii) has a palindromic structure, (iv) is encoded by a low G+C content and (v) contains a highly conserved XerD binding site. However, not all proteobacteria display this dif/XerCD system. Indeed, a sub-group of pathogenic ε-proteobacteria (including Helicobacter sp and Campylobacter sp) harbors a different recombination system, composed of a single recombinase (XerH) which is phylogenetically distinct from the other Xer recombinases and a motif (difH) sharing homologies with difSL. Furthermore, no homologs to dif or Xer recombinases could be detected in small endosymbiont genomes or in certain bacteria with larger chromosomes like the Legionellales. This raises the question of the presence of other chromosomal deconcatenation systems in these species. Our study highlights the complexity of dif/Xer recombinase systems in proteobacteria and paves the way for systematic detection of these components in prokaryotes

Making serine integrases work for us

DNA site-specific recombinases are enzymes (often associated with mobile DNA elements) that catalyse breaking and rejoining of DNA strands at specific points, thereby bringing about precise genetic rearrangements. Serine integrases are a group of recombinases derived from bacteriophages. Their unusual properties, including directionality of recombination and simple site requirements, are leading to their development as efficient, versatile tools for applications in experimental biology, biotechnology, synthetic biology and gene therapy. This article summarizes our current knowledge of serine integrase structure and mechanism, then outlines key factors that affect the performance of these phage recombination systems. Recently published studies, that have expanded the repertoire of available systems and reveal system-specific characteristics, will help us to choose the best integrases for envisaged applications

Methods and compositions for an integrated dual site-specific recombination system for producing environmentally safe and clean transgenic plants

The present invention is directed to the use of multiple site-specific recombination systems for use in transgene containment in plants. More specifically, the present invention describes coordinated excisional DNA recombination by multiple (e.g., dual) recombinases to achieve excision of unwanted transgenic DNA as well as recombinase-coding sequences themselves by self-excision. Further, the simultaneous use of multiple site-specific recombination systems in combination with controllable total sterility technology (i.e., no or reduced sexual reproduction), provides the production of environmentally safe, clean transgenic plants, enhancing the capability and public acceptance of transgenic technology for plant trait modification

Evaluation of the Recombination Efficiencies of FLP Proteins

Site-specific recombination systems are powerful tools for genetic modification. They have been used to integrate a transgene into a pre-defined locus and to remove marker genes from a transgene locus. Two of the most widely used site-specific recombination systems in plants are the Cre/lox system from the bacteriophage P1 and the FLP/FRT system from the yeast Saccharomyces cerevisiae. The Cre/lox system is well-characterized and is the first choice in application of site-specific recombination system. However, some applications such as marker-free site-specific gene integration require the use of two recombination systems. In addition, the availability of alternative recombination systems can offer a flexible choice or the opportunity to develop multiple applications in a single platform. Hence, the FLP/FRT system should be evaluated further for its recombination efficiency, particularly in rice, a model crop plant. Some studies using FLP/FRT systems, with the wild type FLP called FLPwt recombinase, reported low efficiency for regular application of the system in removal of transgenic locus. However, two improved versions of FLPwt: FLPe (thermostable version of FLPwt) and FLPo (mouse-codon optimized version of FLPe) are available and have not been carefully tested in plants. To look for the best choice of FLP recombinase variant in the application of the FLP/FRT system in crop genetic engineering, the relative recombination efficiencies of FLPwt, FLPe and FLPo for marker gene excision from the transgene locus in rice were evaluated. FLPwt, FLPe, and FLPo transgenic rice lines were generated and FLP activity in these lines was evaluated. These experiments revealed that FLPe and FLPo had much higher activity than FLPwt in removing FRT-flanked npt segment to fuse GUS gene with the promoter. These experiments also indicated that FLPo is relatively more efficient than FLPe. Thus, based upon results from the present study, I recommend the use of FLPo in plant genetic engineering

Comparative and Evolutionary Analysis of the Bacterial Homologous Recombination Systems

Homologous recombination is a housekeeping process involved in the maintenance of chromosome integrity and generation of genetic variability. Although detailed biochemical studies have described the mechanism of action of its components in model organisms, there is no recent extensive assessment of this knowledge, using comparative genomics and taking advantage of available experimental data on recombination. Using comparative genomics, we assessed the diversity of recombination processes among bacteria, and simulations suggest that we missed very few homologs. The work included the identification of orthologs and the analysis of their evolutionary history and genomic context. Some genes, for proteins such as RecA, the resolvases, and RecR, were found to be nearly ubiquitous, suggesting that the large majority of bacterial genomes are capable of homologous recombination. Yet many genomes show incomplete sets of presynaptic systems, with RecFOR being more frequent than RecBCD/AddAB. There is a significant pattern of co-occurrence between these systems and antirecombinant proteins such as the ones of mismatch repair and SbcB, but no significant association with nonhomologous end joining, which seems rare in bacteria. Surprisingly, a large number of genomes in which homologous recombination has been reported lack many of the enzymes involved in the presynaptic systems. The lack of obvious correlation between the presence of characterized presynaptic genes and experimental data on the frequency of recombination suggests the existence of still-unknown presynaptic mechanisms in bacteria. It also indicates that, at the moment, the assessment of the intrinsic stability or recombination isolation of bacteria in most cases cannot be inferred from the identification of known recombination proteins in the genomes

The platelet-derived growth factor receptor alpha promoter-directed expression of cre recombinase in mouse placenta.

BackgroundNumerous pathologies of pregnancy originate from placental dysfunction. It is essential to understand the functions of key genes in the placenta in order to discern the etiology of placental pathologies. A paucity of animal models that allow conditional and inducible expression of a target gene in the placenta is a major limitation for studying placental development and function.ResultsTo study the platelet-derived growth factor receptor alpha (PDGFRα)-directed and tamoxifen-induced Cre recombinase expression in the placenta, PDGFRα-CreER mice were crossed with mT/mG dual-fluorescent reporter mice. The expression of endogenous membrane-localized enhanced green fluorescent protein (mEGFP) and/or dTomato in the placenta was examined to identify PDGFRα promoter-directed Cre expression. Pregnant PDGFRα-CreER;mT/mG mice were treated with tamoxifen at various gestational ages. Upon tamoxifen treatment, reporter protein mEGFP was observed in the junctional zone (JZ) and chorionic plate (CP). Furthermore, a single dose of tamoxifen was sufficient to induce the recombination.ConclusionsPDGFRα-CreER expression is restricted to the JZ and CP of mouse placentas. PDGFRα-CreER mice provide a useful tool to conditionally knock out or overexpress a target gene in these regions of the mouse placenta

Phase variation and the Hin protein: in vivo activity measurements, protein overproduction, and purification

The alternate expression of the Salmonella flagellin genes H1 and H2 is controlled by the orientation of a 995-base-pair invertible segment of DNA located at the 5' end of the H2 gene. The hin gene, which is encoded within the invertible region, is essential for the inversion of this DNA segment. We cloned the hin gene into Escherichia coli and placed it under the control of the PL promoter of bacteriophage lambda. These cells overproduced the Hin protein. In vivo inversion activity was measured by using a recombinant lambda phage which contains the H2 and lacZ genes under the control of the invertible region. Using this phage, we showed that the amount of inversion activity is proportional to the amount of Hin protein in the cell. An inactive form of the protein was purified by using the unusual solubility properties of the overproduced protein. The amino acid composition of the protein agreed with the DNA sequence of the hin gene. Antibodies were made to the isolated protein. These antibodies cross-reacted with two other unidentified E. coli proteins

Drip and Mate Operations Acting in Test Tube Systems and Tissue-like P systems

The operations drip and mate considered in (mem)brane computing resemble the operations cut and recombination well known from DNA computing. We here consider sets of vesicles with multisets of objects on their outside membrane interacting by drip and mate in two different setups: in test tube systems, the vesicles may pass from one tube to another one provided they fulfill specific constraints; in tissue-like P systems, the vesicles are immediately passed to specified cells after having undergone a drip or mate operation. In both variants, computational completeness can be obtained, yet with different constraints for the drip and mate operations

VCre/VloxP and SCre/SloxP: new site-specific recombination systems for genome engineering

We developed two new site-specific recombination systems named VCre/VloxP and SCre/SloxP for genome engineering. Their recognition sites are different from Cre recognition sites because VCre and SCre recombinases share less protein similarity with Cre, even though the basic 13-8-13 structures of their recognition sites are identical. Mutant VloxP and SloxP, which have the same uses as mutant loxP, were also developed. VCre/VloxP and SCre/SloxP in combination with Cre/loxP and Flp/FRT systems can serve as powerful tools for genome engineering, especially when used to genetically modify both alleles of a single gene in mouse and human cells