Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection.

Delivery of DNA to cells and its subsequent integration into the host genome is a fundamental task in molecular biology, biotechnology and gene therapy. Here we describe an IP-free one-step method that enables stable genome integration into either prokaryotic or eukaryotic cells. A synthetic mariner transposon is generated by flanking a DNA sequence with short inverted repeats. When purified recombinant Mos1 or Mboumar-9 transposase is co-transfected with transposon-containing plasmid DNA, it penetrates prokaryotic or eukaryotic cells and integrates the target DNA into the genome. In vivo integrations by purified transposase can be achieved by electroporation, chemical transfection or Lipofection of the transposase:DNA mixture, in contrast to other published transposon-based protocols which require electroporation or microinjection. As in other transposome systems, no helper plasmids are required since transposases are not expressed inside the host cells, thus leading to generation of stable cell lines. Since it does not require electroporation or microinjection, this tool has the potential to be applied for automated high-throughput creation of libraries of random integrants for purposes including gene knock-out libraries, screening for optimal integration positions or safe genome locations in different organisms, selection of the highest production of valuable compounds for biotechnology, and sequencing.

Design and synthesis of novel prostaglandin E2 ethanolamide and glycerol ester probes for the putative prostamide receptor(s).

Novel prostaglandin-ethanolamide (PGE2-EA) and glycerol ester (2-PGE2-G) analogs were designed and synthesized to aid in the characterization of a putative prostamide receptor. Our design incorporates the electrophilic isothiocyanato and the photoactivatable azido groups at the terminal tail position of the prototype. Stereoselective Wittig and Horner-Wadsworth-Emmons reactions install the head and the tail moieties of the PGE2 skeleton. The synthesis is completed using Mitsunobu azidation and peptide coupling as the key steps. A chemoenzymatic synthesis for the 2-PGE2-G is described for first time.

Structural Basis for the Inverted Repeat Preferences of mariner Transposases.

The inverted repeat (IR) sequences delimiting the left and right ends of many naturally active mariner DNA transposons are non-identical and have different affinities for their transposase. We have compared the preferences of two active mariner transposases, Mos1 and Mboumar-9, for their imperfect transposon IRs in each step of transposition: DNA binding, DNA cleavage, and DNA strand transfer. A 3.1 Å resolution crystal structure of the Mos1 paired-end complex containing the pre-cleaved left IR sequences reveals the molecular basis for the reduced affinity of the Mos1 transposase DNA-binding domain for the left IR as compared with the right IR. For both Mos1 and Mboumar-9, in vitro DNA transposition is most efficient when the preferred IR sequence is present at both transposon ends. We find that this is due to the higher efficiency of cleavage and strand transfer of the preferred transposon end. We show that the efficiency of Mboumar-9 transposition is improved almost 4-fold by changing the 3' base of the preferred Mboumar-9 IR from guanine to adenine. This preference for adenine at the reactive 3' end for both Mos1 and Mboumar-9 may be a general feature of mariner transposition.

Novel tail and head group prostamide probes.

We report the design and synthesis of novel prostaglandin-ethanolamide (PGE2-EA) analogs containing head and tail group modifications to aid in the characterization of a putative prostamide receptor(s). Our synthetic approach utilizes Horner-Wadsworth-Emmons and Wittig reactions to construct the head and the tail moieties of the key PGE2 precursor, which leads to the final products through a peptide coupling, Swern oxidation and HF/pyridine assisted desilylation. The synthesized analogs were shown not to interact significantly with endocannabinoid proteins and recombinant EP1, EP3 and EP4 receptors and suggest a yet to be identified prostamide receptor as their site(s) of action.

Efficient meltwater drainage through supraglacial streams and rivers on the southwest Greenland ice sheet.

Thermally incised meltwater channels that flow each summer across melt-prone surfaces of the Greenland ice sheet have received little direct study. We use high-resolution WorldView-1/2 satellite mapping and in situ measurements to characterize supraglacial water storage, drainage pattern, and discharge across 6,812 km(2) of southwest Greenland in July 2012, after a record melt event. Efficient surface drainage was routed through 523 high-order stream/river channel networks, all of which terminated in moulins before reaching the ice edge. Low surface water storage (3.6 ± 0.9 cm), negligible impoundment by supraglacial lakes or topographic depressions, and high discharge to moulins (2.54-2.81 cm⋅d(-1)) indicate that the surface drainage system conveyed its own storage volume every <2 d to the bed. Moulin discharges mapped inside ∼52% of the source ice watershed for Isortoq, a major proglacial river, totaled ∼41-98% of observed proglacial discharge, highlighting the importance of supraglacial river drainage to true outflow from the ice edge. However, Isortoq discharges tended lower than runoff simulations from the Modèle Atmosphérique Régional (MAR) regional climate model (0.056-0.112 km(3)⋅d(-1) vs. ∼0.103 km(3)⋅d(-1)), and when integrated over the melt season, totaled just 37-75% of MAR, suggesting nontrivial subglacial water storage even in this melt-prone region of the ice sheet. We conclude that (i) the interior surface of the ice sheet can be efficiently drained under optimal conditions, (ii) that digital elevation models alone cannot fully describe supraglacial drainage and its connection to subglacial systems, and (iii) that predicting outflow from climate models alone, without recognition of subglacial processes, may overestimate true meltwater export from the ice sheet to the ocean.

Biochemical characterization and comparison of two closely related active mariner transposases.

Most DNA transposons move from one genomic location to another by a cut-and-paste mechanism and are useful tools for genomic manipulations. Short inverted repeat (IR) DNA sequences marking each end of the transposon are recognized by a DNA transposase (encoded by the transposon itself). This enzyme cleaves the transposon ends and integrates them at a new genomic location. We report here a comparison of the biophysical and biochemical properties of two closely related and active mariner/Tc1 family DNA transposases: Mboumar-9 and Mos1. We compared the in vitro cleavage activities of the enzymes on their own IR sequences, as well as cross-recognition of their inverted repeat sequences. We found that, like Mos1, untagged recombinant Mboumar-9 transposase is a dimer and forms a stable complex with inverted repeat DNA in the presence of Mg(2+) ions. Mboumar-9 transposase cleaves its inverted repeat DNA in the manner observed for Mos1 transposase. There was minimal cross-recognition of IR sequences between Mos1 and Mboumar-9 transposases, despite these enzymes having 68% identical amino acid sequences. Transposases sharing common biophysical and biochemical properties, but retaining recognition specificity toward their own IR, are a promising platform for the design of chimeric transposases with predicted and improved sequence recognition.

Retrotransposons.

June 27, 1970 was a significant day for our understanding of both the flow of information in biological systems and the evolution of eukaryotic genomes as this was the day that Nature published back-to-back papers reporting the discovery of an enzyme that copies RNA into DNA. This soon became known as reverse transcriptase and the RNA tumour viruses in which it was detected were renamed retroviruses. The realisation that retroviruses can convert their genomic RNA into DNA provided a route by which they could integrate into the chromosomes of infected cells as Howard Temin and his colleagues had proposed some years earlier. At the time it was thought that the ability to copy RNA into DNA would be confined to retroviruses. One of the more startling outcomes of whole genome DNA sequencing has been the discovery that eukaryotes can have more reverse transcriptase genes than genes coding for any other protein, and that the largest single component of many eukaryotic genomes has been generated by reverse transcription.

RNA:RNA interaction can enhance RNA localization in Drosophila oocytes.

RNA localization is a key mechanism for targeting proteins to particular subcellular domains. Sequences necessary and sufficient for localization have been identified, but little is known about factors that affect its kinetics. Transcripts of gurken and the I factor, a non-LTR retrotransposon, colocalize at the nucleus in the dorso-antero corner of the Drosophila oocyte directed by localization signals, the GLS and ILS. I factor RNA localizes faster than gurken after injection into oocytes, due to a difference in the intrinsic localization ability of the GLS and ILS. The kinetics of localization of RNA containing the ILS are enhanced by the presence of a stem-loop, the A loop. This acts as an RNA:RNA interaction element in vivo and in vitro, and stimulates localization of RNA containing other localization signals. RNA:RNA interaction may be a general mechanism for modulating RNA localization and could allow an mRNA that lacks a localization signal to hitchhike on another RNA that has one.

Oogenesis: active heterochromatin.

The genome of Drosophila is protected from DNA damage during oogenesis by a mechanism involving short RNAs. Surprisingly transcription of these RNAs requires that their DNA is associated with a histone modification usually associated with gene silencing.

Formation of polycyclic lactones through a ruthenium-catalyzed ring-closing metathesis/hetero-Pauson-Khand reaction sequence.

Processes that form multiple carbon-carbon bonds in one operation can generate molecular complexity quickly and therefore be used to shorten syntheses of desirable molecules. We selected the hetero-Pauson-Khand (HPK) cycloaddition and ring-closing metathesis (RCM) as two unique carbon-carbon bond-forming reactions that could be united in a tandem ruthenium-catalyzed process. In doing so, complex polycyclic products can be obtained in one reaction vessel from acyclic precursors using a single ruthenium additive that can catalyze sequentially two mechanistically distinct transformations.

Molecular architecture of the Mos1 paired-end complex: the structural basis of DNA transposition in a eukaryote.

A key step in cut-and-paste DNA transposition is the pairing of transposon ends before the element is excised and inserted at a new site in its host genome. Crystallographic analyses of the paired-end complex (PEC) formed from precleaved transposon ends and the transposase of the eukaryotic element Mos1 reveals two parallel ends bound to a dimeric enzyme. The complex has a trans arrangement, with each transposon end recognized by the DNA binding region of one transposase monomer and by the active site of the other monomer. Two additional DNA duplexes in the crystal indicate likely binding sites for flanking DNA. Biochemical data provide support for a model of the target capture complex and identify Arg186 to be critical for target binding. Mixing experiments indicate that a transposase dimer initiates first-strand cleavage and suggest a pathway for PEC formation.

Genome dynamics: transposition and the single cell.

Ciliate development requires assembly of functional genes from segments separated by intervening sequences now shown to have properties of transposons. This may be a relic of a time when transposition drove genome evolution, leading to the differentiation of the germline micronucleus and somatic macronucleus.

A bioinformatics search pipeline, RNA2DSearch, identifies RNA localization elements in Drosophila retrotransposons.

mRNA localization is a widespread mode of delivering proteins to their site of function. The embryonic axes in Drosophila are determined in the oocyte, through Dynein-dependent transport of gurken/TGF-alpha mRNA, containing a small localization signal that assigns its destination. A signal with a similar secondary structure, but lacking significant sequence similarity, is present in the I factor retrotransposon mRNA, also transported by Dynein. It is currently unclear whether other mRNAs exist that are localized to the same site using similar signals. Moreover, searches for other genes containing similar elements have not been possible due to a lack of suitable bioinformatics methods for searches of secondary structure elements and the difficulty of experimentally testing all the possible candidates. We have developed a bioinformatics approach for searching across the genome for small RNA elements that are similar to the secondary structures of particular localization signals. We have uncovered 48 candidates, of which we were able to test 22 for their localization potential using injection assays for Dynein mediated RNA localization. We found that G2 and Jockey transposons each contain a gurken/I factor-like RNA stem-loop required for Dynein-dependent localization to the anterior and dorso-anterior corner of the oocyte. We conclude that I factor, G2, and Jockey are members of a "family" of transposable elements sharing a gurken-like mRNA localization signal and Dynein-dependent mechanism of transport. The bioinformatics pipeline we have developed will have broader utility in fields where small RNA signals play important roles.

RNA interference: endogenous siRNAs derived from transposable elements.

The Piwi-interacting RNA interference pathway plays an important role in suppressing transposable elements in the Drosophila germline. Now, deep sequencing of short RNAs from somatic tissue and cell culture has identified a novel class of endogenous siRNAs that may have a similar role in the soma.

Molecular dissection of Penelope transposable element regulatory machinery.

Penelope-like elements (PLEs) represent a new class of retroelements identified in more than 80 species belonging to at least 10 animal phyla. Penelope isolated from Drosophila virilis is the only known transpositionally active representative of this class. Although the size and structure of the Penelope major transcript has been previously described in both D. virilis and D. melanogaster transgenic strains, the architecture of the Penelope regulatory region remains unknown. In order to determine the localization of presumptive Penelope promoter and enhancer-like elements, segments of the putative Penelope regulatory region were linked to a CAT reporter gene and introduced into D. melanogaster by P-element-mediated transformation. The results obtained using ELISA to measure CAT expression levels and RNA studies, including RT-PCR, suggest that the active Penelope transposon contains an internal promoter similar to the TATA-less promoters of LINEs. The results also suggest that some of the Penelope regulatory sequences control the preferential expression in the ovaries of the adult flies by enhancing expression in the ovary and reducing expression in the carcass. The possible significance of the intron within Penelope for the function and evolution of PLEs, and the effect of Penelope insertions on adjacent genes, are discussed.

Crystallization of a Mos1 transposase-inverted-repeat DNA complex: biochemical and preliminary crystallographic analyses.

A complex formed between Mos1 transposase and its inverted-repeat DNA has been crystallized. The crystals diffract to 3.25 A resolution and exhibit monoclinic (P2(1)) symmetry, with unit-cell parameters a = 120.8, b = 85.1, c = 131.6 A, beta = 99.3 degrees . The X-ray diffraction data display noncrystallographic twofold symmetry and characteristic dsDNA diffraction at approximately 3.3 A. Biochemical analyses confirmed the presence of DNA and full-length protein in the crystals. The relationship between the axis of noncrystallographic symmetry, the unit-cell axes and the DNA diffraction pattern are discussed. The data are consistent with the previously proposed model of the paired-ends complex containing a dimer of the transposase.

Live imaging of Drosophila gonad formation reveals roles for Six4 in regulating germline and somatic cell migration.

BACKGROUND: Movement of cells, either as amoeboid individuals or in organised groups, is a key feature of organ formation. Both modes of migration occur during Drosophila embryonic gonad development, which therefore provides a paradigm for understanding the contribution of these processes to organ morphogenesis. Gonads of Drosophila are formed from three distinct cell types: primordial germ cells (PGCs), somatic gonadal precursors (SGPs), and in males, male-specific somatic gonadal precursors (msSGPs). These originate in distinct locations and migrate to associate in two intermingled clusters which then compact to form the spherical primitive gonads. PGC movements are well studied, but much less is known of the migratory events and other interactions undergone by their somatic partners. These appear to move in organised groups like, for example, lateral line cells in zebra fish or Drosophila ovarian border cells. RESULTS: We have used time-lapse fluorescence imaging to characterise gonadal cell behaviour in wild type and mutant embryos. We show that the homeodomain transcription factor Six4 is required for the migration of the PGCs and the msSGPs towards the SGPs. We have identified a likely cause of this in the case of PGCs as we have found that Six4 is required for expression of Hmgcr which codes for HMGCoA reductase and is necessary for attraction of PGCs by SGPs. Six4 affects msSGP migration by a different pathway as these move normally in Hmgcr mutant embryos. Additionally, embryos lacking fully functional Six4 show a novel phenotype in which the SGPs, which originate in distinct clusters, fail to coalesce to form unified gonads. CONCLUSION: Our work establishes the Drosophila gonad as a model system for the analysis of coordinated cell migrations and morphogenesis using live imaging and demonstrates that Six4 is a key regulator of somatic cell function during gonadogenesis. Our data suggest that the initial association of SGP clusters is under distinct control from the movements that drive gonad compaction.

Preparation of aliphatic ketones through a ruthenium-catalyzed tandem cross-metathesis/allylic alcohol isomerization.

Grubbs' 2nd generation and Hoveyda-Grubbs' ruthenium alkylidenes are shown to be effective catalysts for cross-metatheses of allylic alcohols with cyclic and acyclic olefins, as well as isomerization of the resulting allylic alcohols to alkyl ketones. The net result of this new tandem methodology is a single-flask process that provides highly functionalized, ketone-containing products from simple allylic alcohol precursors. [reaction: see text]

D-six4 plays a key role in patterning cell identities deriving from the Drosophila mesoderm.

Patterning of the Drosophila embryonic mesoderm requires the regulation of cell type-specific factors in response to dorsoventral and anteroposterior axis information. For the dorsoventral axis, the homeodomain gene, tinman, is a key patterning mediator for dorsal mesodermal fates like the heart. However, equivalent mediators for more ventral fates are unknown. We show that D-six4, which encodes a Six family transcription factor, is required for the appropriate development of most cell types deriving from the non-dorsal mesoderm - the fat body, somatic cells of the gonad, and a specific subset of somatic muscles. Misexpression analysis suggests that D-Six4 and its likely cofactor, Eyes absent, are sufficient to impose these fates on other mesodermal cells. At stage 10, the mesodermal expression patterns of D-six4 and tin are complementary, being restricted to the dorsal and non-dorsal regions respectively. Our data suggest that D-six4 is a key mesodermal patterning mediator at this stage that regulates a variety of cell-type-specific factors and hence plays an equivalent role to tin. At stage 9, however, D-six4 and tin are both expressed pan-mesodermally. At this stage, tin function is required for full D-six4 expression. This may explain the known requirement for tin in some non-dorsal cell types.