Integrity Of Backbone Research Articles

Optimized Agrobacterium-mediated sorghum transformation protocol and molecular data of transgenic sorghum plants.

Agrobacterium-mediated sorghum transformation frequency has been enhanced significantly via medium optimization using immature embryos from sorghum variety TX430 as the target tissue. The new transformation protocol includes the addition of elevated copper sulfate and 6-benzylaminopurine in the resting and selection media. Using Agrobacterium strain LBA4404, the transformation frequency reached over 10% using either of two different selection marker genes, moPAT or PMI, and any of three different vectors in large-scale transformation experiments. With Agrobacterium strain AGL1, the transformation frequencies were as high as 33%. Using quantitative PCR analyses of 1,182 T0 transgenic plants representing 675 independent transgenic events, data was collected for T-DNA copy number, intact or truncated T-DNA integration, and vector backbone integration into the sorghum genome. A comparison of the transformation frequencies and molecular data characterizing T-DNA integration patterns in the transgenic plants derived from LBA4404 versus AGL1 transformation revealed that twice as many transgenic high-quality events were generated when AGL1 was used compared to LBA4404. This is the first report providing molecular data for T-DNA integration patterns in a large number of independent transgenic plants in sorghum.Electronic supplementary materialThe online version of this article (doi:10.1007/s11627-013-9583-z) contains supplementary material, which is available to authorized users.

A chemical perspective on transcriptional fidelity: dominant contributions of sugar integrity revealed by unlocked nucleic acids.

Transcription unlocked: A synthetic chemical biology approach involving unlocked nucleic acids was used to dissect the contribution of sugar backbone integrity to the RNA Polymerase II (Pol II) transcription process. An unexpected dominant role for sugar-ring integrity in Pol II transcriptional efficiency and fidelity was revealed.

Transformation of apple (Malus × domestica) using mutants of apple acetolactate synthase as a selectable marker and analysis of the T-DNA integration sites

Apple acetolactate synthase mutants were generated by site-specific mutagenesis and successfully used as selection marker in tobacco and apple transformation. T-DNA/Apple genome junctions were analysed using genome-walking PCR and sequencing. An Agrobacterium-mediated genetic transformation system was developed for apple (Malus × domestica), using mutants of apple acetolactate synthase (ALS) as a selectable marker. Four apple ALS mutants were generated by site-specific mutagenesis and subsequently cloned under the transcriptional control of the CaMV 35S promoter and ocs 3' terminator, in a pART27-derived plant transformation vector. Three of the four mutations were found to confer resistance to the herbicide Glean(®), containing the active agent chlorsulfuron, in tobacco (Nicotiana tabacum) transformation. In apple transformation, leaf explants infected with Agrobacterium tumefaciens EHA105 containing one of the three ALS mutants resulted in the production of shoots on medium containing 2-8 μg L(-1) Glean(®), whilst uninfected wild-type explants failed to regenerate shoots or survive on medium containing 1 and 3 μg L(-1) Glean(®), respectively. Glean(®)-resistant, regenerated shoots were further multiplied and rooted on medium containing 10 μg L(-1) Glean(®). The T-DNA and apple genome-DNA junctions from eight rooted transgenic apple plants were analysed using genome-walking PCR amplification and sequencing. This analysis confirmed T-DNA integration into the apple genome, identified the genome integration sites and revealed the extent of any vector backbone integration, T-DNA rearrangements and deletions of apple genome DNA at the sites of integration.

The impact of Production Tax Credits on the profitable production of electricity from wind in the U.S.

A spatial financial model using wind data derived from assimilated meteorological condition was developed to investigate the profitability and competitiveness of onshore wind power in the contiguous U.S. It considers not only the resulting estimated capacity factors for hypothetical wind farms but also the geographically differentiated costs of local grid connection. The levelized cost of wind-generated electricity for the contiguous U.S. is evaluated assuming subsidy levels from the Production Tax Credit (PTC) varying from 0 to 4 ¢/kWh under three cost scenarios: a reference case, a high cost case, and a low cost case. The analysis indicates that in the reference scenario, current PTC subsidies of 2.1 ¢/kWh are at a critical level in determining the competitiveness of wind-generated electricity compared to conventional power generation in local power market. Results from this study suggest that the potential for profitable wind power with the current PTC subsidy amounts to more than seven times existing demand for electricity in the entire U.S. Understanding the challenges involved in scaling up wind energy requires further study of the external costs associated with improvement of the backbone transmission network and integration into the power grid of the variable electricity generated from wind.

Backbone-free transformation of barrel medic (Medicago truncatula) with a Medicago-derived transfer DNA

In the present work, Agrobacterium tumefaciens-mediated genetic transformation of the model legume Medicago truncatula Gaertn. (barrel medic) was carried out using the pSIM843 vector that contains a Medicago-derived transfer DNA, delineated by a 25-bp sequence homologous to bacterial T-DNA borders. The transfer DNA contains an expression cassette for the nptII (neomycin phosphotransferase) gene and is flanked by an expression cassette for the backbone integration marker gene ipt (isopentenyl transferase). Our results demonstrate that the Medicago-derived RB-like elements efficiently support DNA mobilization from A. tumefaciens to M. truncatula. Kanamycin-resistant shoots with normal phenotype and ipt-shooty lines were recovered at a frequency of 11.7 and 7.8%, respectively. Polymerase chain reaction (PCR) analyses demonstrated that 44.4% of the independent transgenic lines were backbone-free and evidenced the occurrence of backbone-transfer events.

Elimination of the Plasmid Bacterial Backbone in Site-Directed Transgenesis

For cellular and animal transgenesis, FLP- and Cre-recombinase gene capture systems are highly effective to provide stable integration of a donor plasmid carrying the transgene cassette of interest into an engineered genomic locus in a given cell line. However, in many protocols, the entire plasmid bacterial backbone is integrated along with the transgene cassette. Here, we present a very simple yet highly efficient method for excluding plasmid bacterial backbone integration. The transgene cassette, including a single FLP recognition target site, is specifically amplified by PCR, and the resulting DNA ligated into minicircles can serve as donor DNA in FLP-mediated recombination. Interestingly, the elimination of the bacterial backbone increased expression of the inserted transgene. The presented method is simple and efficient for generating transgene cassette insertions devoid of the bacterial backbone.

Charge transport in DNA molecules: Cooperative interplay between the disordered base-pair channel and the ordered backbone

Charge transport in DNA molecules has raised considerable interest because of its importance in biological processes and potential applications in nanoscale devices. A DNA molecule can be viewed as a quasi-one-dimensional system composed of stacked base pairs (AT , CG) together with backbones of sugar phosphates. Motivated by recent experimental observations on the importance of the backbone integrity, we investigate the interplay between charge transport through the ordered backbone and disordered base stacks with random sequences. By analytical and numerical calculations, we find that the coupling between the backbone and base-pair channels plays an important role in charge transport. The backbone can generate effective hopping constants well beyond the adjacent base pairs, enhancing charge transport through the base-pair channel. The corresponding enhancement of the localization length is nearly independent of the length of the DNA and increases with increasing coupling between backbone and base pair. Our model can explain qualitatively several experimental observations.

Evidence of genotype dependency within Agrobacterium tumefaciens in relation to the integration of vector backbone sequence in transgenic Phytophthora infestans-tolerant potato

In this study the effect of Agrobacterium tumefaciens genotype of two strains AGL1 and LBA4404 was investigated in regard to the propensity for backbone integration during the transformation of potato for blight tolerance conferred by the resistant to blight (RB) gene carried by the vector pCLDO4541. A PCR based walking approach was employed to identify left and right backbone sequences as well as for selected genes carried on the plasmid backbone. It was found that adjacent to the left border insertion site, the integration of backbone sequence was greater for AGL1 than for LBA4404; however, the opposite was observed with regards to the right border T-DNA junction. Considering both T-DNA borders LBA4404 was found to have a two fold greater integration potential for backbone than the AGL1. The possibility of only backbone integration in T-DNA negative plants was also investigated with the average rate of integration between the two strains calculated at 4.2% with LBA4404 recording a three fold greater occurrence of backbone integration than AGL1. In summary, evidence of Agrobacterium genotype dependency showed that LBA4404 has greater potential to integrate non-T-DNA vector sequence than AGL1 and this should be taken into account when utilising the listed A. tumefaciens genotypes in generating transgenic potato. Additionally, the application of a PCR and primer walking system proved to be reliable and allows for fine detailed studies of backbone sequence integration of transgenic plant.

Cytokinin vectors mediate marker-free and backbone-free plant transformation

Conventional Agrobacterium-mediated transformation methods rely on complex and genotype-specific tissue culture media for selection, proliferation, and regeneration of genetically modified cells. Resulting transgenic plants may not only contain selectable marker genes but also carry fragments of the vector backbone. Here, we describe a new method for the production of transgenic plants that lack such foreign DNA. This method employs vectors containing the bacterial isopentenyltransferase (ipt) gene as backbone integration marker. Agrobacterium strains carrying the resulting ipt gene-containing "cytokinin" vectors were used to infect explants of various Solanaceous plant species as well as canola (Brassica napus). Upon transfer to hormone-free media, 1.8% to 9.9% of the infected explants produced shoots that contained a marker-free T-DNA while lacking the backbone integration marker. These frequencies often equal or exceed those for backbone-free conventional transformation.

Characterisation of T-DNA loci and vector backbone sequences in transgenic wheat produced by Agrobacterium-mediated transformation

Detailed molecular characterisation of transgene loci is a requirement for gaining regulatory approval for environmental release of genetically modified crops. In cereals, it is generally accepted that Agrobacterium-mediated transformation generates cleaner transgene loci with lower copy number and fewer rearrangements than those generated by biolistics. However, in wheat there has been little detailed analysis of T-DNA insertions at genetic and molecular level. Wheat lines transformed using Agrobacterium tumefaciens with bar and gusA (GUS) genes were subjected to genetic and molecular analysis. Unlike previous studies of transgene loci in wheat, we used functional assays for PAT and GUS proteins, combined with PCR and Southern analysis to detect the presence, copy number, linkage and transmission of two transgenes inserted in the same T-DNA. Thirty-four independent transgenic lines were categorised into three types: type I events (38% of total) where the gusA and bar genes displayed complete genetic linkage, segregating together as a single functional locus at the expected ratio of 3:1; type II events (18%), which possessed two or more transgene loci each containing gusA and bar; and type III events (44%), containing an incomplete T-DNA in which either the gusA or bar gene was lost. Most lines in this last category had lost the bar gene situated near the left T-DNA border. Southern analysis indicated that 30% of all lines possessed a single T-DNA copy containing gusA and bar. However, when data on expression and molecular analysis are combined, only 23% of all lines have single copy T-DNAs in which both gene cassettes are functioning. We also report on the presence of plasmid backbone DNA sequence in transgene loci detected using primer pairs outside the left and right T-DNA borders and within the plasmid selectable marker (NptI) gene. Approximately two thirds of the lines contained some vector backbone DNA, more frequently adjacent to the left border. Taken together, these data imply unstable left border function causing premature T-strand termination or read-through into vector backbone. As far as we are aware, this is the first report revealing near border T-DNA truncation and vector backbone integration in wheat transgenic lines produced by Agrobacterium-mediated transformation.

Molecular analysis of transgene and vector backbone integration into the barley genome following Agrobacterium-mediated transformation

We report a large-scale study on the frequency of transgene and T-DNA backbone integration following Agrobacterium-mediated transformation of immature barley embryos. One hundred and ninety-one plant lines were regenerated after hygromycin selection and visual selection for GFP expression at the callus stage. Southern blotting performed on a subset of 53 lines that were PCR positive for the GFP gene documented the integration of the GFP gene in 27 of the lines. Twenty-three of these lines expressed GFP in T(1) plantlets. Southern blotting with a vector backbone probe revealed that 13 of the 27 lines possessed one or more vector backbone fragments illustrating the regular occurrence of vector backbone integration following Agrobacterium infection of barley immature embryos.

Characterization of T-DNA integration in creeping bentgrass

Agrobacterium-mediated transformation provides a powerful tool for plant biotechnology. Great efforts have been made to unravel the underlying mechanisms of T-DNA integration. Creeping bentgrass ( Agrostis palustris Huds.), an economically important turfgrass species, has been widely studied for genetic transformation. In order to characterize T-DNA integration in this species, we analyzed T-DNA junction sequences in transgenic bentgrass obtained by transformation with A. tumefaciens strain EHA101 carrying a pPZP201-derived plasmid. Using T-DNA specific primers, inverse polymerase chain reaction (IPCR) and standard PCR were performed to identify T-DNA/plant junctions and T-DNA/T-DNA junctions, respectively. Sequence comparisons revealed that T-DNA right border (RB) termini were more intact, represented by a smaller range of border deletions and no backbone integration, compared to T-DNA left border (LB) termini. Multiple T-DNA arrays inserted at a single chromosome locus were detected in various configurations. It was shown that the formation of the multiple T-DNA arrays was governed by illegitimate recombination (IR), with sequence microsimilarities over and/or close to the T-DNA breaking points. It was also demonstrated that the fusion of two T-DNA copies was frequently associated with the formation of filler DNA and significant T-DNA truncation. Compositional analysis over the bentgrass sequences revealed a similar A/ T ( A + T) content but a polarized distribution of A and T associated with LB flanking sequence tags (FSTs) and RB FSTs, which was further documented in Arabidopsis T-DNA FSTs. DNA folding analysis on T-DNA FSTs suggested that the T-DNA target region had a potential of forming secondary structures, which may designate an active region for T-DNA integration. The results presented in this study shed new light on microsimilarity-based illegitimate recombination and advance our understanding of T-DNA integration.

Crop improvement through modification of the plant's own genome.

Plant genetic engineering has, until now, relied on the incorporation of foreign DNA into plant genomes. Public concern about the extent to which transgenic crops differ from their traditionally bred counterparts has resulted in molecular strategies and gene choices that limit, but not eliminate, the introduction of foreign DNA. Here, we demonstrate that a plant-derived (P-) DNA fragment can be used to replace the universally employed Agrobacterium transfer (T-) DNA. Marker-free P-DNAs are transferred to plant cell nuclei together with conventional T-DNAs carrying a selectable marker gene. By subsequently linking a positive selection for temporary marker gene expression to a negative selection against marker gene integration, 29% of derived regeneration events contain P-DNA insertions but lack any copies of the T-DNA. Further refinements are accomplished by employing Omega-mutated virD2 and isopentenyl transferase cytokinin genes to impair T-DNA integration and select against backbone integration, respectively. The presented methods are used to produce hundreds of marker-free and backbone-free potato (Solanum tuberosum) plants displaying reduced expression of a tuber-specific polyphenol oxidase gene in potato. The modified plants represent the first example of genetically engineered plants that only contain native DNA.

Controlled release of the fibronectin central cell binding domain from polymeric microspheres

Non-ionic surfactants have been employed as alternatives to PVA for the emulsification–encapsulation of a conformationally labile protein (FIII9′-10) into PLGA microspheres. FIII9′-10 was encapsulated using a w/o/w double emulsification–evaporation technique and the microspheres fabricated were characterized by SEM and CLSM. The peptide backbone integrity of FIII9′-10 was assayed by SDS-PAGE and the degree of unfolding of FIII9′-10 following emulsification–encapsulation was assessed using a fibroblast cell-attachment assay. The encapsulation efficiency for FIII9′-10 was 25% when using PVA, compared to 50–60% when using Igepal CA-630 or Triton-X100, with values below for the other surfactants. FIII9′-10 released from microspheres promoted cell attachment in a concentration-dependent manner, only Igepal CA-630 and Triton X-100 maintaining near-maximal cell attachment, indicating that the conformation of the relatively unstable FIII9′ domain was preserved. All non-ionic surfactants reduced microsphere surface porosity, compared to PVA, and an increasing surface rugosity (leading to minor ‘ridges’) could be correlated with decreasing surfactant HLB. Low surface porosities did not effect the diffusion of FIII9′-10 from the microspheres' internal pores in a ‘burst release’, as may have been imagined. In summary, non-ionic surfactants should be considered over PVA for the maintenance of biological activity of conformationally labile proteins during encapsulation.

Cell Biology of the Human Thiamine Transporter-1 (hTHTR1): INTRACELLULAR TRAFFICKING AND MEMBRANE TARGETING MECHANISMS

The human thiamine transporter hTHTR1 is involved in the cellular accumulation of thiamine (vitamin B1) in many tissues. Thiamine deficiency disorders, such as thiamine-responsive megaloblastic anemia (TRMA), which is associated with specific mutations within hTHTR1, likely impairs the functionality and/or intracellular targeting of hTHTR1. Unfortunately, nothing is known about the mechanisms that control the intracellular trafficking or membrane targeting of hTHTR1. To identify molecular determinants involved in hTHTR1 targeting, we generated a series of hTHTR1 truncations fused with the green fluorescent protein and imaged the targeting and trafficking dynamics of each construct in living duodenal epithelial cells. Whereas the full-length fusion protein was functionally expressed at the plasma membrane, analysis of the truncated mutants demonstrated an essential role for both NH(2)-terminal sequence and the integrity of the backbone polypeptide for cell surface expression. Most notably, truncation of hTHTR1 within a region where several TRMA truncations are clustered resulted in intracellular retention of the mutant protein. Finally, confocal imaging of the dynamics of intracellular hTHTR1 vesicles revealed a critical role for microtubules, but not microfilaments, in hTHTR1 trafficking. Taken together, these results correlate hTHTR1 structure with cellular expression profile and reveal a critical dependence on hTHTR1 backbone integrity and microtubule-based trafficking processes for functional expression of hTHTR1.