Resolution of Holliday junctions by eukaryotic DNA topoisomerase I.

The Holliday junction, a key intermediate in both homologous and site-specific recombination, is generated by the reciprocal exchange of single strands between two DNA duplexes. Resolution of the junctions can occur in two directions with respect to flanking markers, either restoring the parental DNA configuration or generating a genetic crossover. Recombination can be regulated, in principle, by factors that influence the directionality of the resolution step. We demonstrate that the vaccinia virus DNA topoisomerase, a eukaryotic type I enzyme, catalyzes resolution of synthetic Holliday junctions in vitro. The mechanism entails concerted transesterifications at two recognition sites, 5'-CCCTT decreases, that are opposed within a partially mobile four-way junction. Cruciforms are resolved unidirectionally and with high efficiency into two linear duplexes. These findings suggest a model whereby type I topoisomerases may either promote or suppress genetic recombination in vivo.

The penicillin gene cluster is amplified in tandem repeats linked by conserved hexanucleotide sequences.

The penicillin biosynthetic genes (pcbAB, pcbC, penDE) of Penicillium chrysogenum AS-P-78 were located in a 106.5-kb DNA region that is amplified in tandem repeats (five or six copies) linked by conserved TTTACA sequences. The wild-type strains P. chrysogenum NRRL 1951 and Penicillium notatum ATCC 9478 (Fleming's isolate) contain a single copy of the 106.5-kb region. This region was bordered by the same TTTACA hexanucleotide found between tandem repeats in strain AS-P-78. A penicillin overproducer strain, P. chrysogenum E1, contains a large number of copies in tandem of a 57.9-kb DNA fragment, linked by the same hexanucleotide or its reverse complementary TGTAAA sequence. The deletion mutant P. chrysogenum npe10 showed a deletion of 57.9 kb that corresponds exactly to the DNA fragment that is amplified in E1. The conserved hexanucleotide sequence was reconstituted at the deletion site. The amplification has occurred within a single chromosome (chromosome I). The tandem reiteration and deletion appear to arise by mutation-induced site-specific recombination at the conserved hexanucleotide sequences.

Ins1 (Cre) knock-in mice for beta cell-specific gene recombination.

AIMS/HYPOTHESIS: Pancreatic beta cells play a central role in the control of glucose homeostasis by secreting insulin to stimulate glucose uptake by peripheral tissues. Understanding the molecular mechanisms that control beta cell function and plasticity has critical implications for the pathophysiology and therapy of major forms of diabetes. Selective gene inactivation in pancreatic beta cells, using the Cre-lox system, is a powerful approach to assess the role of particular genes in beta cells and their impact on whole body glucose homeostasis. Several Cre recombinase (Cre) deleter mice have been established to allow inactivation of genes in beta cells, but many show non-specific recombination in other cell types, often in the brain. METHODS: We describe the generation of Ins1 (Cre) and Ins1 (CreERT2) mice in which the Cre or Cre-oestrogen receptor fusion protein (CreERT2) recombinases have been introduced at the initiation codon of the Ins1 gene. RESULTS: We show that Ins1 (Cre) mice induce efficient and selective recombination of floxed genes in beta cells from the time of birth, with no recombination in the central nervous system. These mice have normal body weight and glucose homeostasis. Furthermore, we show that tamoxifen treatment of adult Ins1 (CreERT2) mice crossed with Rosa26-tdTomato mice induces efficient recombination in beta cells. CONCLUSIONS/INTERPRETATION: These two strains of deleter mice are useful new resources to investigate the molecular physiology of pancreatic beta cells.

Manipulation of adenovirus early region 1 rescue plasmids by homologous recombination in Saccharomyces cerevisiae

The manipulation of large (>10 kb) plasmid systems amplifies problems common to traditional cloning strategies. Unique or rare restriction enzyme recognition sequences are uncommon and very rarely located in opportunistic locations. Making site-specific deletions and insertions in larger plasmids consequently leads to multiple step cloning strategies that are often limited by time-consuming, low efficiency linker insertions or blunt-end cloning strategies. Manipulation ofthe adenovirus genome and the genomes ofother viruses as bacterial plasmids are systems that typify such situations. Recombinational cloning techniques based on homologous recombination in Saccharomyces cerevisiae that circumvent many ofthese common problems have been developed. However, these techniques are rarely realistic options for such large plasmid systems due to the above mentioned difficulties associated with the addition ofrequired yeast DNA replication, partitioning and selectable marker sequences. To determine ifrecombinational cloning techniques could be modified to simplify the manipulation of such a large plasmid system, a recombinational cloning system for the creation of human adenovirus EI-deletion rescue plasmids was developed. Here we report for the first time that the 1,456 bp TRP1/ARS fragment ofYRp7 is alone sufficient to foster successful recombinational cloning without additional partitioning sequences, using only slight modifications of existing protocols. In addition, we describe conditions for efficient recombinational cloning involving simultaneous deletion of large segments ofDNA (>4.2 kb) and insertion of donor fragment DNA using only a single non-unique restriction site. The discovery that recombinational cloning can foster large deletions has been used to develop a novel recombiliational cloillng technique, selectable inarker 'kilockouf" recombinational cloning, that uses deletion of a yeast selectable marker coupled with simultaneous negative and positive selection to reduce background transformants to undetectable levels. The modification of existing protocols as described in this report facilitates the use of recombinational cloning strategies that are otherwise difficult or impractical for use with large plasmid systems. Improvement of general recombinational cloning strategies and strategies specific to the manipulation ofthe adenovirus genome are considered in light of data presented herein.

Tid1/Rdh54 promotes colocalization of Rad51 and Dmc1 during meiotic recombination

Two RecA homologs, Rad51 and Dmc1, assemble as cytologically visible complexes (foci) at the same sites on meiotic chromosomes. Time course analysis confirms that co-foci appear and disappear as the single predominant form. A large fraction of co-foci are eliminated in a red1 mutant, which is expected as a characteristic of the interhomolog-specific recombination pathway. Previous studies suggested that normal Dmc1 loading depends on Rad51. We show here that a mutation in TID1/RDH54, encoding a RAD54 homolog, reduces Rad51-Dmc1 colocalization relative to WT. A rad54 mutation, in contrast, has relatively little effect on RecA homolog foci except when strains also contain a tid1/rdh54 mutation. The role of Tid1/Rdh54 in coordinating RecA homolog assembly may be very direct, because Tid1/Rdh54 is known to physically bind both Dmc1 and Rad51. Also, Dmc1 foci appear early in a tid1/rdh54 mutant. Thus, Tid1 may normally act with Rad51 to promote ordered RecA homolog assembly by blocking Dmc1 until Rad51 is present. Finally, whereas double-staining foci predominate in WT nuclei, a subset of nuclei with expanded chromatin exhibit individual Rad51 and Dmc1 foci side-by-side, suggesting that a Rad51 homo-oligomer and a Dmc1 homo-oligomer assemble next to one another at the site of a single double-strand break (DSB) recombination intermediate.

Targeted DNA recombination in vivo using an adenovirus carrying the cre recombinase gene.

Conditional gene expression and gene deletion are important experimental approaches for examining the functions of particular gene products in development and disease. The cre-loxP system from bacteriophage P1 has been used in transgenic animals to induce site-specific DNA recombination leading to gene activation or deletion. To regulate the recombination in a spatiotemporally controlled manner, we constructed a recombinant adenoviral vector, Adv/cre, that contained the cre recombinase gene under regulation of the herpes simplex virus thymidine kinase promoter. The efficacy and target specificity of this vector in mediating loxP-dependent recombination were analyzed in mice that had been genetically engineered to contain loxP sites in their genome. After intravenous injection of the Adv/cre vector into adult animals, the liver and spleen showed the highest infectivity of the adenovirus as well as the highest levels of recombination, whereas other tissues such as kidney, lung, and heart had lower levels of infection and recombination. Only trace levels of recombination were detected in the brain. However, when the Adv/cre vector was injected directly into specific regions of the adult brain, including the cerebral cortex, hippocampus, and cerebellum, recombination was detectable at the injection site. Furthermore, when the Adv/cre vector was injected into the forebrains of neonatal mice, the rearranged toxP locus from recombination could be detected in the injected regions for at least 8 weeks. Taken together, these results demonstrate that the Adv/cre vector expressing a functional cre protein is capable of mediating loxP-dependent recombination in various tissues and the recombined gene locus may in some cases be maintained for an extended period. The use of the adenovirus vector expressing cre combined with localized delivery to specific tissues may provide an efficient means to achieve conditional gene expression or knockout with precise spatiotemporal control.

Covalent protein-DNA complexes at the 5' strand termini of meiosis-specific double-strand breaks in yeast.

During meiosis in Saccharomyces cerevisiae, the first chemical step in homologous recombination is the occurrence of site-specific DNA double-strand breaks (DSBs). In wild-type cells, these breaks undergo resection of their 5' strand termini to yield molecules with 3' single-stranded tails. We have further characterized the breaks that accumulate in rad50S mutant stains defective in DSB resection. We find that these DSBs are tightly associated with protein via what appears to be a covalent linkage. When genomic DNA is prepared from meiotic rad50S cultures without protease treatment steps, the restriction fragments diagnostic of DSBs selectively partition to the organic-aqueous interphase in phenol extractions and band at lower than normal density in CsCl density gradients. Selective partitioning and decreased buoyant density are abolished if the DNA is treated with proteinase K prior to analysis. Similar results are obtained with sae2-1 mutant strains, which have phenotypes identical to rad50S mutants. The protein is bound specifically to the 5' strand termini of DSBs and is present at both 5' ends in at least a fraction of breaks. The stability of the complex to various protein denaturants and the strand specificity of the attachment are most consistent with a covalent linkage to DSB termini. We propose that the DSB-associated protein is the catalytic subunit of the meiotic recombination initiation nuclease and that it cleaves DNA via a covalent protein-DNA intermediate.

Incorporating a TEV cleavage site reduces the solubility of nine recombinant mouse proteins

Failure to express soluble proteins in bacteria is mainly attributed to the properties of the target protein itself, as well as the choice of the vector, the purification tag and the linker between the tag and protein, and codon usage. The expression of proteins with fusion tags to facilitate subsequent purification steps is a widely used procedure in the production of recombinant proteins. However, the additional residues can affect the properties of the protein; therefore, it is often desirable to remove the tag after purification. This is usually done by engineering a cleavage site between the tag and the encoded protein that is recognised by a site-specific protease, such as the one from tobacco etch virus (TEV). In this study, we investigated the effect of four different tags on the bacterial expression and solubility of nine mouse proteins. Two of the four engineered constructs contained hexahistidine tags with either a long or short linker. The other two constructs contained a TEV cleavage site engineered into the linker region. Our data show that inclusion of the TEV recognition site directly downstream of the recombination site of the Invitrogen Gateway vector resulted, in a loss of solubility of the nine mouse proteins. Our work suggests that one needs to be very careful when making modifications to expression vectors and combining different affinity and fusion tags and cleavage sites: (c) 2006 Elsevier Inc. All rights reserved.

Miscibility and specific interactions in blends of poly(4-vinylphenol) and poly(2-ethoxyethyl methacrylate)

The properties of the hydrogen-bonded polymer blends of poly(4-vinylphenol) and poly(2-ethoxyethyl methacrylate) are presented. Spectroscopic techniques such as C-13 solid-state NMR and FT-IR are used to probe specific interactions of the blends at various compositions. Spectral features from both techniques revealed that site-specific interactions are present, consistent with a significant degree of mixing of the blend components. Changes in chemical shift and line shape of the phenolic carbon and carbonyl resonances in the C-13 CPMAS spectra of the blends as a function of composition are interpreted as resulting from changes in the relative intensities of two closely overlapped signals. A quantitative measure of hydrogen-bonded carbonyl groups using C-13 NMR has been obtained which agreed well with the results from FT-IR analyses. It is also shown that C-13 NMR can be used to measure the fraction of hydroxyl groups associated with carbonyl groups, which was not possible previously using FT-IR due to extensive overlapping of bands in the hydroxyl stretching region. The results of measurements of H-1 T-1 and 1H T-1 rho indicate that PVPh and PEEMA are intimately mixed on a scale less than 2-3 nm.

In Vitro Methods for Specific IgE Detection on Cow’s Milk Allergy

Background: A new method for determining serum specific IgE (IMMULITE“ 2000 3gAllergy) has recently become available. Objective: To evaluate the clinical performance of IMMULITE 2000 in the diagnosis of cow’s milk allergy compared with that of UniCAP“. Additionally, we verified the behavior of both methods at two diagnostic decision points proposed by other authors. Methods: The study population consisted of 31 children with cow’s milk allergy (group A) and a control group of 19 atopic children without food allergy(group B). A blood sample from each child was tested using both methods and the results were compared. Results: In group A, the values for cow’s milk IgE ranged from 0.35 kU/L (the lowest common detection limit) to above 100 kU/L. In group B, the values were less than 1.1 kU/L for IMMULITE 2000 and less than 1.6 kU/L for UniCAP. An agreement of 90 % in IgE classes was obtained. Both methods demonstrated exactly the same diagnostic performance(sensitivity: 100 %; specificity: 78.9 %; negative predictive value: 100%; positive predictive value: 84.6%;efficiency: 90.2 %). The evaluation of the two methods at the two different decision points proposed in the literature showed a better positive predictive value with UniCAP, but we obtained equivalent performance with IMMULITE 2000 by choosing higher cutoff values. Conclusions: We conclude that IMMULITE 2000 is as effective as UniCAP in the diagnosis of cow’s milk allergy. Both methods can be used to obtain site-specific decision points that are population, age and disease dependent.

An analysis of the use of photogrammetric sorting to audit construction and demolition waste production on site

This study analyses the area of construction and demolition waste (C & D W) auditing. The production of C&DW has grown year after year since the Environmental Protection Agency (EPA) first published a report in 1996 which provided data for C&D W quantities for 1995 (EPA, 1996a). The most recent report produced by the EPA is based on data for 2005 (EPA, 2006). This report estimated that the quantity of C&DW produced for that period to be 14 931 486 tonnes. However, this is a ‘data update’ report containing an update on certain waste statistics so any total provided would not be a true reflection of the waste produced for that period. This illustrates that a more construction site-specific form of data is required. The Department of Building and Civil Engineering in the Galway-Mayo Institute of Technology have carried out two recent research projects (Grimes, 2005; Kelly, 2006) in this area, which have produced waste production indicators based on site-specific data. This involved the design and testing of an original auditing tool based on visual characterisation and the application of conversion factors. One of the main recommendations of these studies was to compare this visual characterisation approach with a photogrammetric sorting methodology. This study investigates the application of photogrammetric sorting on a residential construction site in the Galway region. A visual characterisation study is also carried out on the same project to compare the two methodologies and assess the practical application in a construction site environment. Data collected from the waste management contractor on site was also used to provide further evaluation. From this, a set of waste production indicators for new residential construction was produced: □ 50.8 kg/m2 for new residential construction using data provided by the visual characterisation method and the Landfill Levy conversion factors. □ 43 kg/m2 for new residential construction using data provided by the photogrammetric sorting method and the Landfill Levy conversion factors. □ 23.8 kg/m2 for new residential construction using data provided by Waste Management Contractor (WMC). The acquisition of the data from the waste management contractor was a key element for testing of the information produced by the visual characterisation and photogrammetric sorting methods. The actual weight provided by the waste management contractor shows a significant difference between the quantities provided.

A New Large-DNA-Fragment Delivery System Based on Integrase Activity from an Integrative and Conjugative Element.

During the past few decades, numerous plasmid vectors have been developed for cloning, gene expression analysis, and genetic engineering. Cloning procedures typically rely on PCR amplification, DNA fragment restriction digestion, recovery, and ligation, but increasingly, procedures are being developed to assemble large synthetic DNAs. In this study, we developed a new gene delivery system using the integrase activity of an integrative and conjugative element (ICE). The advantage of the integrase-based delivery is that it can stably introduce a large DNA fragment (at least 75 kb) into one or more specific sites (the gene for glycine-accepting tRNA) on a target chromosome. Integrase recombination activity in Escherichia coli is kept low by using a synthetic hybrid promoter, which, however, is unleashed in the final target host, forcing the integration of the construct. Upon integration, the system is again silenced. Two variants with different genetic features were produced, one in the form of a cloning vector in E. coli and the other as a mini-transposable element by which large DNA constructs assembled in E. coli can be tagged with the integrase gene. We confirmed that the system could successfully introduce cosmid and bacterial artificial chromosome (BAC) DNAs from E. coli into the chromosome of Pseudomonas putida in a site-specific manner. The integrase delivery system works in concert with existing vector systems and could thus be a powerful tool for synthetic constructions of new metabolic pathways in a variety of host bacteria.

RecA-mediated annealing of single-stranded DNA and its relation to the mechanism of homologous recombination.

We demonstrate that RecA protein can mediate annealing of complementary DNA strands in vitro by at least two different mechanisms. The first annealing mechanism predominates under conditions where RecA protein causes coaggregation of single-stranded DNA (ssDNA) molecules and where RecA-free ssDNA stretches are present on both reaction partners. Under these conditions annealing can take place between locally concentrated protein-free complementary sequences. Other DNA aggregating agents like histone H1 or ethanol stimulate annealing by the same mechanism. The second mechanism of RecA-mediated annealing of complementary DNA strands is best manifested when preformed saturated RecA-ssDNA complexes interact with protein-free ssDNA. In this case, annealing can occur between the ssDNA strand resident in the complex and the ssDNA strand that interacts with the preformed RecA-ssDNA complex. Here, the action of RecA protein reflects its specific recombination promoting mechanism. This mechanism enables DNA molecules resident in the presynaptic RecA-DNA complexes to be exposed for hydrogen bond formation with DNA molecules contacting the presynaptic RecA-DNA filament.

Functional analysis of the post-transcriptional regulator RsmA reveals a novel RNA-binding site.

The RsmA family of RNA-binding proteins are global post-transcriptional regulators that mediate extensive changes in gene expression in bacteria. They bind to, and affect the translation rate of target mRNAs, a function that is further modulated by one or more, small, untranslated competitive regulatory RNAs. To gain new insights into the nature of this protein/RNA interaction, we used X-ray crystallography to solve the structure of the Yersinia enterocolitica RsmA homologue. RsmA consists of a dimeric beta barrel from which two alpha helices are projected. From structure-based alignments of the RsmA protein family from diverse bacteria, we identified key amino acid residues likely to be involved in RNA-binding. Site-specific mutagenesis revealed that arginine at position 44, located at the N terminus of the alpha helix is essential for biological activity in vivo and RNA-binding in vitro. Mutation of this site affects swarming motility, exoenzyme and secondary metabolite production in the human pathogen Pseudomonas aeruginosa, carbon metabolism in Escherichia coli, and hydrogen cyanide production in the plant beneficial strain Pseudomonas fluorescens CHA0. R44A mutants are also unable to interact with the small untranslated RNA, RsmZ. Thus, although possessing a motif similar to the KH domain of some eukaryotic RNA-binding proteins, RsmA differs substantially and incorporates a novel class of RNA-binding site.