Parallel In Vivo DNA Assembly by Recombination: Experimental Demonstration and Theoretical Approaches

The development of synthetic biology requires rapid batch construction of large gene networks from combinations of smaller units. Despite the availability of computational predictions for well-characterized enzymes, the optimization of most synthetic biology projects requires combinational constructions and tests. A new building-brick-style parallel DNA assembly framework for simple and flexible batch construction is presented here. It is based on robust recombination steps and allows a variety of DNA assembly techniques to be organized for complex constructions (with or without scars). The assembly of five DNA fragments into a host genome was performed as an experimental demonstration.     

Strand bias influences the mechanism of gene editing directed by single-stranded DNA oligonucleotides

Gene editing directed by modified single-stranded DNA oligonucleotides has been used to alter a single base pair in a variety of biological systems. It is likely that gene editing is facilitated by the direct incorporation of the oligonucleotides via replication and/or by direct conversion, most likely through the DNA mismatch repair pathway. The phenomenon of strand bias, however, as well as its importance to the gene editing reaction itself, has yet to be elucidated in terms of mechanism. We have taken a reductionist approach by using a genetic readout in Eschericha coli and a plasmid-based selectable system to evaluate the influence of strand bias on the mechanism of gene editing. We show that oligonucleotides (ODNs) designed to anneal to the lagging strand generate 100-fold greater 'editing' efficiency than 'those that anneal to' the leading strand. The majority of editing events (～70%) occur by the incorporation of the ODN during replication within the lagging strand. Conversely, ODNs that anneal to the leading strand generate fewer editing events although this event may follow either the incorporation or direct conversion pathway. In general, the influence of DNA replication is independent of which ODN is used suggesting that the importance of strand bias is a reflection of the underlying mechanism used to carry out gene editing.     

Molecular regulation of CCN2 in the intervertebral disc: Lessons learned from other connective tissues

Connective tissue growth factor (CCN2/CTGF) plays an important role in extracellular matrix synthesis, especially in skeletal tissues such as cartilage, bone, and the intervertebral disc. As a result there is a growing interest in examining the function and regulation of this important molecule in the disc. This review discusses the regulation of CCN2 by TGF-β and hypoxia, two critical determinants that characterize the disc microenvironment, and discusses known functions of CCN2 in the disc. The almost ubiquitous regulation of CCN2 by TGF-β, including that seen in the disc, emphasizes the importance of the TGF-β–CCN2 relationship, especially in terms of extracellular matrix synthesis. Likewise, the unique cross-talk between CCN2 and HIF-1 in the disc highlights the tissue and niche specific mode of regulation. Taken together the current literature supports an anabolic role for CCN2 in the disc and its involvement in the maintenance of tissue homeostasis during both health and disease. Further studies of CCN2 in this tissue may reveal valuable targets for the biological therapy of disc degeneration.     

Trimethylsilyloxo complexes of oxomolybdenum(V)

The complexes LMo^VIO₂X [L?=?hydrotris(3,5-dimethylpyrazol-1-yl)borate; X?=?Cl, Br, NCS, OPh, SPh, SCH₂Ph] are converted to air-stable complexes LMo^VO(OSiMe₃)X by one-electron coupled electron-electrophile transfer (CEET) reactions involving cobaltocene and the electrophilic reagent Me₃SiCl. These complexes may also be obtained from LMo^VO(OH)X by reaction with Me₃SiCl in the presence of base. LMo^VO(OSiMe₃)(SCH₂Ph) crystallises in space group P2₁/n, with a?=?8.526 (1) Å, b?=?23.141 (3) Å, c?=?16.499 (2) Å, β?=?103.75 (12)° and Z?=?4. The complex exhibits a distorted octahedral structure with a facially tridentate L ligand and mutually cis oxo [Mo=O?=?1.675 (4) Å], silyloxo [Mo–O?=?1.932 (4) Å] and thiolato [Mo–S?=?2.398 (2) Å] ligands. The detailed redox properties of LMo^VO(OR)X (R?=?SiMe₃, alkyl, aryl) differ from those of LMo^VO(OH)X. Centres [Mo^VO(OR)] are candidates for the stable "inhibited" forms of certain molybdenum enzymes formed under conditions which apparently disfavour the catalytically active [Mo^VO(OH)] centres. In the coordinating solvent pyridine (py), both LMo^VIO₂(SPh) and LMo^VO(OSiMe₃)(SPh) are reduced in one-electron steps to stable LMo^IVO(py)(SPh). LMo^IVO(py)(SR) complexes are also obtained from LMo^VIO₂(SR) (R?=?Ph, CH₂Ph, CHMe₂) via a two-electron oxygen atom transfer reaction with tertiary phosphines in pyridine. Consequently, the Mo(IV) product is accessible via a concerted two-electron step or via two one-electron steps.     

Considerations that affect optimised simulation in a running jump for height

This study used a computer simulation model to investigate various considerations that affect optimum peak height in a running jump. A planar eight-segment computer simulation model with extensor and flexor torque generators at five joints was formulated and customised to an elite male high jumper. A simulation was matched to a recorded high jumping performance by varying the activation profiles of each of the torque generators giving a simulated peak height of 1.99m compared to the recorded performance of 2.01 m. In order to maximise the peak height reached by the mass centre in the flight phase, the activation profiles were varied, keeping the same initial conditions as in the matching simulation. Optimisations were carried out without any constraints, with constraints on the angular momentum at take-off, with further constraints on joint angles, and with additional requirements of robustness to perturbations of activation timings. A peak height of 2.37 m was achieved in the optimisation without constraints. Introducing the three constraints in turn resulted in peak heights of 2.21, 2.14 and 1.99m. With all three types of constraints included, the peak height was similar to that achieved in the recorded performance. It is concluded that such considerations have a substantial influence on optimum technique and must be included in studies using optimised simulations.     

The impact of biochemistry vs. population membership on floral scent profiles in colour polymorphic Hesperis matronalis

Background and Aims

Studies of floral scent evolution often attribute variation in floral scent to differences in pollinator behaviour, ignoring the potential for shared biochemistry between floral scent and floral colour to dictate patterns of phenotypic variation in scent production. To determine the relative effects of shared biochemistry and/or localized population-level phenomena on floral scent phenotype, floral scent composition and emission rate were examined in five wild populations of colour polymorphic Hesperis matronalis (Brassicaceae).

Methods

Floral scent was collected by in situ dynamic headspace extraction on purple and white colour morphs in each of five wild populations. Gas chromatography–mass spectroscopy of extracts allowed determination of floral scent composition and emission rate for all individuals, which were examined by non-metric multidimensional scaling and analysis of variance (ANOVA), respectively, to determine the contributions of floral colour and population membership to scent profile variation.

Key Results

Despite the fact that colour morph means were very similar in some populations and quite different in other populations, colour morphs within populations did not differ from each other in terms of scent composition or emission rate. Populations differed significantly from one another in terms of both floral scent composition and emission rate.

Conclusions

Shared biochemistry alone cannot explain the variation in floral scent phenotype found for H. matronalis. Such a result may suggest that the biochemical association between floral scent and floral colour is complex or dependent on genetic background. Floral scent does vary significantly with population membership; several factors, including environmental conditions, founder effects and genetics, may account for this differentiation and should be considered in future studies.Key words: Hesperis matronalis, floral scent, floral colour, plant volatiles, population differentiation, scent composition, scent emission rate, terpenoids, aromatics     

Genome scale assessment of a species translocation program

Despite increased use of species translocations, controversy remains regarding the efficacy and efficiency of the strategy in obtaining conservation goals. Much of this controversy results from vague program objectives, unclear definitions of success, and lack of follow-up monitoring. We used the translocation program initiated by Zoo Knoxville for the federally threatened Bog Turtle (Glyptemys muhlenbergii) as a case study to demonstrate how genomic assessments not only assess the success of program objectives, but also allow managers to quickly obtain baseline data from which program objectives and explicit definitions of ‘success’ can be determined. Here we used 7030 SNP markers derived from RADseq data to confirm the premise that different source populations are genetically differentiated. Then we tested whether the release population has enhanced genetic diversity, as expected from a deliberate admixture. Although the release population had greater diversity than any source population, variation was lower than expected from modeling admixture with equal source contribution. Our results support the premise that genetic diversity can be maximized by including representatives from as many natural populations as possible. But failure to achieve the expected level of diversity could result from nonrandom success of founders from different sources or unrecorded bias in the implementation of the release program. Many existing and future translocation programs would benefit from genetic assessment similar to that conducted here with Bog Turtles.     

Ion channelopathies and migraine pathogenesis

Migraine is a common neurological disorder that affects approximately 12–20% of the general adult population. Migraine pathogenesis is complex and not wholly understood. Molecular genetic investigations, imaging and biochemical studies, have unveiled a number of interconnected neurological pathways which seem to have a cause and effect component integral to its cause. Much weight of migraine attack initiation can be placed on the initial trigger and the pathways involved in its neuronal counter reaction. Ion channels play a large role in the generation, portrayal and mitigation of the brains response to external triggers. Several genetic studies have identified and implicated a number of ion channelopathy genes which may contribute to this generalised process. This review will focus on the genetics of migraine with particular emphasis placed on the potentially important role genes HEPH (responsible for iron transport and homeostasis) and KCNK18 (important for the transport and homeostasis of potassium) play in migraine cause.     

Plastid division is mediated by combinatorial assembly of plastid division proteins

Plastids arise by division from pre-existing organelles, and with the recent characterization of several new components of plastid division our understanding of the division process in higher plants has improved dramatically. However, it is still not known how these different protein components act together during division. Here we analyse protein-protein interactions between all known stromal plastid division proteins. Using a combination of quantitative yeast two-hybrid assays, in planta co-localization studies, fluorescence resonance energy transfer and bimolecular fluorescence complementation assays we show that these proteins do not act in isolation but rather in protein complexes to govern appropriate plastid division. We have previously shown that AtMinD1 forms functional homodimers and we show here that in addition to homodimerization AtMinD1 also interacts with AtMinE1. Furthermore, AtMinE1 has the ability to homodimerize. We also demonstrate that proteins from both FtsZ families (AtFtsZ1-1 and AtFtsZ2-1) not only interact with themselves but also with each other, and we show that these interactions are not dependent on correct Z-ring formation. Further to this we demonstrate that ARC6 specifically interacts with the core domain of AtFtsZ2-1, but not with AtFtsZ1-1, providing in planta evidence for a functional difference between the two FtsZ protein families in plants. Our studies have enabled us to construct a meaningful intraplastidic protein-protein interaction map of all known stromal plastid division proteins in Arabidopsis.     

Chemically defined medium and <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Background  

C. elegans has been established as a powerful genetic system. Use of a chemically defined medium (C. elegans Maintenance Medium (CeMM)) now allows standardization and systematic manipulation of the nutrients that animals receive. Liquid cultivation allows automated culturing and experimentation and should be of use in large-scale growth and screening of animals.