Solution conformations of early intermediates in Mos1 transposition

DNA transposases facilitate genome rearrangements by moving DNA transposons around and between genomes by a cut-and-paste mechanism. DNA transposition proceeds in an ordered series of nucleoprotein complexes that coordinate pairing and cleavage of the transposon ends and integration of the cleaved ends at a new genomic site. Transposition is initiated by transposase recognition of the inverted repeat sequences marking each transposon end. Using a combination of solution scattering and biochemical techniques, we have determined the solution conformations and stoichiometries of DNA-free Mos1 transposase and of the transposase bound to a single transposon end. We show that Mos1 transposase is an elongated homodimer in the absence of DNA and that the N-terminal 55 residues, containing the first helix-turn-helix motif, are required for dimerization. This arrangement is remarkably different from the compact, crossed architecture of the dimer in the Mos1 paired-end complex (PEC). The transposase remains elongated when bound to a single-transposon end in a pre-cleavage complex, and the DNA is bound predominantly to one transposase monomer. We propose that a conformational change in the single-end complex, involving rotation of one half of the transposase along with binding of a second transposon end, could facilitate PEC assembly

The proteins encoded by the pogo-like Lemi1 element bind the TIRs and subterminal repeated motifs of the Arabidopsis Emigrant MITE: consequences for the transposition mechanism of MITEs

MITEs (miniature inverted-repeated transposable elements) are a particular class of defective DNA transposons usually present within genomes as high copy number populations of highly homogeneous elements. Although an active MITE, the mPing element, has recently been characterized in rice, the transposition mechanism of MITEs remains unknown. It has been proposed that transposases of related transposons could mobilize MITEs in trans. Moreover, it has also been proposed that the presence of conserved terminal inverted-repeated (TIR) sequences could be the only requirement of MITEs for mobilization, allowing divergent or unrelated elements to be mobilized by a particular transposase. We present here evidence for a recent mobility of the Arabidopsis Emigrant MITE and we report on the capacity of the proteins encoded by the related Lemi1 transposon, a pogo-related element, to specifically bind Emigrant elements. This suggests that Lemi1 could mobilize Emigrant elements and makes the Lemi1/Emigrant couple an ideal system to study the transposition mechanism of MITEs. Our results show that Lemi1 proteins bind Emigrant TIRs but also bind cooperatively to subterminal repeated motifs. The requirement of internal sequences for the formation of proper DNA/protein structure could affect the capacity of divergent MITEs to be mobilized by distantly related transposases

IS911 transpososome assembly as analysed by tethered particle motion

Initiation of transposition requires formation of a synaptic complex between both transposon ends and the transposase (Tpase), the enzyme which catalyses DNA cleavage and strand transfer and which ensures transposon mobility. We have used a single-molecule approach, tethered particle motion (TPM), to observe binding of a Tpase derivative, OrfAB[149], amputated for its C-terminal catalytic domain, to DNA molecules carrying one or two IS911 ends. Binding of OrfAB[149] to a single IS911 end provoked a small shortening of the DNA. This is consistent with a DNA bend introduced by protein binding to a single end. This was confirmed using a classic gel retardation assay with circularly permuted DNA substrates. When two ends were present on the tethered DNA in their natural, inverted, configuration, Tpase not only provoked the short reduction in length but also generated species with greatly reduce effective length consistent with DNA looping between the ends. Once formed, this ‘looped’ species was very stable. Kinetic analysis in real-time suggested that passage from the bound unlooped to the looped state could involve another species of intermediate length in which both transposon ends are bound. DNA carrying directly repeated ends also gave rise to the looped species but the level of the intermediate species was significantly enhanced. Its accumulation could reflect a less favourable synapse formation from this configuration than for the inverted ends. This is compatible with a model in which Tpase binds separately to and bends each end (the intermediate species) and protein–protein interactions then lead to synapsis (the looped species)

Challenges and Opportunities in Finfish Nutrition

Much of the criticism leveled at aquaculture (e.g., dependency on animal-derived feedstuffs, nutrient-laden effluent discharges, and increased organic contamination in edible products) can be traced to the feeds in use. Accordingly, finfish nutritionists are being challenged to formulate feeds that not only meet the nutritional requirements of livestock but also minimize production costs, limit environmental impacts, and enhance product quality. These challenges not only add considerable complexity to finfish nutrition but also afford opportunities to avoid some of the mistakes made by other industries in the past. From a review of the current status of finfish nutrition with respect to major nutrient classes, we comment on future opportunities and promising avenues of research. Alternative protein sources, specifically those derived from marine bycatch, plants, and microbes, are discussed, as well as methods to facilitate their implementation in finfish feeds. Dietary lipid, its role in fish bioenergetics and physiology, and quality of aquaculture products is reviewed with special emphasis on alternative lipid sources and finishing diets. Carbohydrates and fiber are discussed in terms of nutrient-sparing, least-cost diet formulation and digestive physiology. Micronutrients are reviewed in terms of current knowledge of requirements and, along with other dietary immunostimulants, are given further consideration in a review of nutriceuticals and application in finfish feeds. The status of nutritional research in new aquaculture species is also outlined. By integrating classical approaches with emerging technologies, dietary formulations, and species, finfish nutritionists may identify means to increase production efficiency and sustainability and provide for the continued success of aquaculture

Structural role of the flanking DNA in mariner transposon excision

During cut-and-paste mariner/Tc1 transposition, transposon DNA is cut precisely at its junction with flanking DNA, ensuring the transposon is neither shortened nor lengthened with each transposition event. Each transposon end is flanked by a TpA dinucleotide: the signature target site duplication of mariner/Tc1 transposition. To establish the role of this sequence in accurate DNA cleavage, we have determined the crystal structure of a pre-second strand cleavage mariner Mos1 transpososome. The structure reveals the route of an intact DNA strand through the transposase active site before second strand cleavage. The crossed architecture of this pre-second strand cleavage paired-end complex supports our proposal that second strand cleavage occurs in trans. The conserved mariner transposase WVPHEL and YSPDL motifs position the strand for accurate DNA cleavage. Base-specific recognition of the flanking DNA by conserved amino acids is revealed, defining a new role for the WVPHEL motif in mariner transposition and providing a molecular explanation for in vitro mutagenesis data. Comparison of the pre-TS cleavage and post-cleavage Mos1 transpososomes with structures of Prototype Foamy Virus intasomes suggests a binding mode for target DNA prior to Mos1 transposon integration

Clinical Predictors of Immune Reconstitution following Combination Antiretroviral Therapy in Patients from the Australian HIV Observational Database

A small but significant number of patients do not achieve CD4 T-cell counts >500 cells/µl despite years of suppressive cART. These patients remain at risk of AIDS and non-AIDS defining illnesses. The aim of this study was to identify clinical factors associated with CD4 T-cell recovery following long-term cART.Patients with the following inclusion criteria were selected from the Australian HIV Observational Database (AHOD): cART as their first regimen initiated at CD4 T-cell count <500 cells/µl, HIV RNA<500 copies/ml after 6 months of cART and sustained for at least 12 months. The Cox proportional hazards model was used to identify determinants associated with time to achieve CD4 T-cell counts >500 cells/µl and >200 cells/µl.501 patients were eligible for inclusion from AHOD (n = 2853). The median (IQR) age and baseline CD4 T-cell counts were 39 (32-47) years and 236 (130-350) cells/µl, respectively. A major strength of this study is the long follow-up duration, median (IQR) = 6.5(3-10) years. Most patients (80%) achieved CD4 T-cell counts >500 cells/µl, but in 8%, this took >5 years. Among the patients who failed to reach a CD4 T-cell count >500 cells/µl, 16% received cART for >10 years. In a multivariate analysis, faster time to achieve a CD4 T-cell count >500 cells/µl was associated with higher baseline CD4 T-cell counts (p<0.001), younger age (p = 0.019) and treatment initiation with a protease inhibitor (PI)-based regimen (vs. non-nucleoside reverse transcriptase inhibitor, NNRTI; p = 0.043). Factors associated with achieving CD4 T-cell counts >200 cells/µl included higher baseline CD4 T-cell count (p<0.001), not having a prior AIDS-defining illness (p = 0.018) and higher baseline HIV RNA (p<0.001).The time taken to achieve a CD4 T-cell count >500 cells/µl despite long-term cART is prolonged in a subset of patients in AHOD. Starting cART early with a PI-based regimen (vs. NNRTI-based regimen) is associated with more rapid recovery of a CD4 T-cell count >500 cells/µl