Hybridization Partners Research Articles

Detection of feral GT73 transgenic oilseed rape (Brassica napus) along railway lines on entry routes to oilseed factories in Switzerland

To obtain a reference status prior to cultivation of genetically modified oilseed rape (OSR, Brassica napus L.) in Switzerland, the occurrence of feral OSR was monitored along transportation routes and at processing sites. The focus was set on the detection of (transgenic) OSR along railway lines from the Swiss borders with Italy and France to the respective oilseed processing factories in Southern and Northern Switzerland (Ticino and region of Basel). A monitoring concept was developed to identify sites of largest risk of escape of genetically modified plants into the environment in Switzerland. Transport spillage of OSR seeds from railway goods cars particularly at risk hot spots such as switch yards and (un)loading points but also incidental and continuous spillage were considered. All OSR plants, including their hybridization partners which were collected at the respective monitoring sites were analyzed for the presence of transgenes by real-time PCR. On sampling lengths each of 4.2 and 5.7km, respectively, 461 and 1,574 plants were sampled in Ticino and the region of Basel. OSR plants were found most frequently along the routes to the oilseed facilities, and in larger amounts on risk hot spots compared to sites of random sampling. At three locations in both monitored regions, transgenic B. napus line GT73 carrying the glyphosate resistance transgenes gox and CP4 epsps were detected (Ticino, 22 plants; in the region of Basel, 159).

Altering Colloidal Surface Functionalization Using DNA Encapsulated Inside Monodisperse Gelatin Microsphere Templates

Soluble oligonucleotides are typically introduced to bulk solution to promote hybridization activity on DNA-functionalized surfaces. Here, an alternative approach is explored by encapsulating secondary target strands inside semipermeable colloidal satellite assemblies, then triggering their release at 37 °C for subsequent surface hybridization activity. To prepare DNA-loaded satellite assemblies, uniform gelatin microspheres are fabricated using microfluidics, loaded with 15 base-long secondary DNA targets, capped with a polyelectrolyte bilayer, and finally coated with a monolayer of polystyrene microspheres functionalized with duplexes comprised of immobilized probes and soluble, 13 base-long hybridization partner strands. Once warmed to 37 °C, secondary DNA targets are released from the gelatin template and then competitively displace the shorter, original hybridization partners on the polystyrene microspheres.

Measuring in Situ Primary and Competitive DNA Hybridization Activity on Microspheres

Microspheres serve as convenient substrates for studying DNA activity on surfaces. Here, in addition to employing conventional sample preparation involving multiple wash and resuspension steps prior to flow cytometry measurements, we also directly sampled the reaction volume to acquire in situ measurements of primary and competitive hybridization events. Even in the absence of post hybridization wash steps, nonspecific binding events were negligible and thus allowed for direct, quantitative assessment of hybridization events as they occurred on colloidal surfaces. The in situ results indicate that primary duplex formation between immobilized probes and soluble targets on microsphere surfaces is less favorable than predicted by solution models. The kinetics of competitive displacement of primary hybridization partners by secondary targets measured in situ or post washing also deviate from expectations based on theoretical solution thermodynamics, but are consistent with predicted kinetic trends stemming from differences in either the toehold base length or branch migration.

Simulating evolutionary responses of an introgressed insect resistance trait for ecological effect assessment of transgene flow: a model for supporting informed decision-making in environmental risk assessment.

Predicting outcomes of transgene flow from arable crops requires a system perspective that considers ecological and evolutionary processes within a landscape context. In Europe, the arable weed Raphanus raphanistrum is a potential hybridization partner of oilseed rape, and the two species are ecologically linked through the common herbivores Meligethes spp. Observations in Switzerland show that high densities of Meligethes beetles maintained by oilseed rape crops can lead to considerable damage on R. raphanistrum. We asked how increased insect resistance in R. raphanistrum – as might be acquired through introgression from transgenic oilseed rape – would affect seed production under natural herbivore pressure. In simulation experiments, plants protected against Meligethes beetles produced about twice as many seeds as unprotected plants. All stages in the development of reproductive structures from buds to pods were negatively affected by the herbivore, with the transition from buds to flowers being the most vulnerable. We conclude that resistance to Meligethes beetles could confer a considerable selective advantage upon R. raphanistrum in regions where oilseed rape is widely grown.

Exploring locked nucleic acids as a bio-inspired materials assembly and disassembly tool

Oligonucleotides hold great promise as a recognition-based biomaterials assembly and disassembly tool. Chemically modified oligonucleotides such as locked nucleic acids (LNA) provide the added advantage of nuclease resistance. In the current study, we focus on programming the assembly and disassembly of LNA-linked colloidal particles as a function of sequence composition. We find that incorporation of LNA residues (∼30%) into either one or both primary hybridization partner strands results in a higher duplex density than for isosequential DNA strands. Mismatched primary hybridization partners with sequence length of 11–15 bases have similar initial primary duplex densities. The displacement of mismatched strands by 15 base-long, perfectly matched competitive target strands, however, does depend on the base length of the original mismatched partner strand. Confocal microscopy confirms that substantial colloidal assembly occurs for both perfectly matched and mismatched LNA sequences that are 9 bases in length. Extensive disassembly for the mismatched case is then triggered through the introduction of 15 base-long competitive target strands. Our work demonstrates that LNA can be used to programmatically assemble and disassemble colloidal particles.

Immunotherapy using allogeneic squamous cell tumor–dendritic cell fusion hybrids

Tumor-associated antigens (TAAs) are known to be immunotherapy targets; thus tumor-sharing TAA may be used as a fusion hybrid partner to confer protection against subsequent tumor challenge. The squamous cell carcinomas (SCCs), SCCVII and B4B8, were used in C3H/HEN mice: SCCVII (H-2(k)) is syngeneic, B4B8 (H-2(d)) is allogeneic. Experiments using tumor alone included hyperimmunization schedule, subdermal and intranodal routes. Mice were challenged 2 weeks later. Fusion hybrids were created from both SCC tumor cell lines and syngeneic dendritic cells (DCs). These were delivered intranodally for immunization, and mice were challenged with tumor 2 weeks later. Only syngeneic tumor given subdermally was able to protect after tumor challenge 2 weeks later. Hyperimmunization schedule did not alter these findings. However, fusion hybrid immunization from both allogeneic and syngeneic SCCs conferred protection after tumor challenge. Allogeneic tumor-DC fusion hybrids targeting TAA can protect against subsequent tumor challenge.

Effects of mismatches on DNA as an isothermal assembly and disassembly tool

Recently, our group developed an approach to drive disassembly of DNA-linked structures at a fixed temperature by adding soluble secondary targets that competitively displace the shorter, original hybridization partners. Here, instead of relying solely on base length differences between primary and secondary targets to drive isothermal disassembly, we introduce a single base mismatch to further weaken the DNA linkages holding colloidal particles together and promote subsequent disassembly upon the addition of perfectly-matched secondary targets.

Optimization of the BLASTN substitution matrix for prediction of non-specific DNA microarray hybridization

DNA microarray measurements are susceptible to error caused by non-specific hybridization between a probe and a target (cross-hybridization), or between two targets (bulk-hybridization). Search algorithms such as BLASTN can quickly identify potentially hybridizing sequences. We set out to improve BLASTN accuracy by modifying the substitution matrix and gap penalties. We generated gene expression microarray data for samples in which 1 or 10% of the target mass was an exogenous spike of known sequence. We found that the 10% spike induced 2-fold intensity changes in 3% of the probes, two-third of which were decreases in intensity likely caused by bulk-hybridization. These changes were correlated with similarity between the spike and probe sequences. Interestingly, even very weak similarities tended to induce a change in probe intensity with the 10% spike. Using this data, we optimized the BLASTN substitution matrix to more accurately identify probes susceptible to non-specific hybridization with the spike. Relative to the default substitution matrix, the optimized matrix features a decreased score for A–T base pairs relative to G–C base pairs, resulting in a 5–15% increase in area under the ROC curve for identifying affected probes. This optimized matrix may be useful in the design of microarray probes, and in other BLASTN-based searches for hybridization partners.

Reversing DNA-Mediated Adhesion at a Fixed Temperature

Recognition-based assembly of micron- to nano-sized colloidal particles functionalized with DNA has generated great interest in the past decade; however, reversing the assembly process is typically achieved by thermal denaturation of the oligonucleotide duplexes. Here, we report an alternative disassembly approach at a fixed temperature using competitive hybridization events between immobilized and soluble oligonucleotide strands. Microspheres are first aggregated via primary hybridization events between immobilized DNA strands with a weak, but sufficient, affinity for partner strands to link complementary surfaces together. To reverse the aggregation process, soluble oligonucleotides are then added to competitively displace the original hybridization partners through secondary hybridization events. Using flow cytometry to quantify hybridization events and microscopy to examine DNA-mediated aggregation and redispersion, we found that the efficiency of competitive displacement is based upon (1) the difference in base pair matches between the primary and secondary target for the same probe sequence and (2) the concentration of hybridizing oligonucleotides participating in microsphere aggregation. To the best of our knowledge, this study is the first to employ DNA hybridization events to mediate reversible adhesion between colloidal particles at a fixed temperature.

Conformational and Chiral Selection of Oligonucleotides

In view of a better understanding of chiral selection of oligonucleotides, we have studied the hybridization of D- and L-CNA (cyclohexane nucleic acids) and D- and L-DNA, with chiral D-beta-homo-DNA and achiral PNA (peptide nucleic acids). PNA hybridizes as well with D-DNA, L-DNA as with D-beta-homo-DNA. The structure of the PNA x D-beta-homo-DNA complex is different from the PNA x DNA duplexes. D-CNA prefers D-DNA as hybridization partner, while L-CNA prefers D-beta-homo-DNA as hybridization partner. The conformation of the enantiomeric oligonucleotides D-CNA and L-CNA in the supramolecular complex with D-DNA and D-beta-homo-DNA, respectively, is different. These data may contribute to the confirmation of a hypothesis of the existence of achiral informative polymers as RNA predecessor, and to the understanding of homochirality of nucleic acids.

Efficient identification of DNA hybridization partners in a sequence database

The specific hybridization of complementary DNA molecules underlies many widely used molecular biology assays, including the polymerase chain reaction and various types of microarray analysis. In order for such an assay to work well, the primer or probe must bind to its intended target, without also binding to additional sequences in the reaction mixture. For any given probe or primer, potential non-specific binding partners can be identified using state-of-the-art models of DNA binding stability. Unfortunately, these models rely on dynamic programming algorithms that are too slow to apply on a genomic scale. We present an algorithm that efficiently scans a DNA database for short (approximately 20-30 base) sequences that will bind to a query sequence. We use a filtering approach, in which a series of increasingly stringent filters is applied to a set of candidate k-mers. The k-mers that pass all filters are then located in the sequence database using a precomputed index, and an accurate model of DNA binding stability is applied to the sequence surrounding each of the k-mer occurrences. This approach reduces the time to identify all binding partners for a given DNA sequence in human genomic DNA by approximately three orders of magnitude, from two days for the ENCODE regions to less than one minute for typical queries. Our approach is scalable to large DNA sequences. Our method can scan the human genome for medium strength binding sites to a candidate PCR primer in an average of 34.5 minutes. Software implementing the algorithms described here is available at http://noble.gs.washington.edu/proj/dna-binding.

Multiple hybrid formation in natural populations: concerted evolution of the internal transcribed spacer of nuclear ribosomal DNA (ITS) in North American Arabis divaricarpa (Brassicaceae).

DNA sequence variation of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA from Arabis holboellii, A. drummondii, and its putative hybrid A. divaricarpa was analyzed to study hybrid speciation in a species system geographically covering nearly the entire North American continent. Based on molecular systematics the investigated species are better combined under the genus Boechera. Multiple intraindividual ITS copies were detected in numerous accessions of A. divaricarpa, and, to a minor extent, in the parental taxa. Comparative phylogenetic analysis demonstrates that reticulate evolution is common. Consequently, concerted evolution of ITS regions resulted in different types of ITS fragments not only in hybrid populations but also in one of the parental taxa, A. holboellii. Hybrid formation often occurred independently at different sites and at different times, which is reflected by ITS copies resampling the original parental sequence variation in different ways. Some biogeographic structuring of genetic diversity is apparent and mirrors postglacial migration routes. Hybridization, reticulation, and apomixis are assumed to be the major forces driving speciation processes in this species complex. Analysis of conserved regions and secondary structures of the ITS region provided no evidence that, in this system, hybrid ITS evolution is predominantly driven in a particular direction. However, two regions in the ITS1 and ITS2, respectively, show higher mutation rates than expected from outgroup comparisons. Strong evidence for the occurrence of apomixis in A. holboellii and A. divaricarpa has come from pollen size measurements and estimations of pollen quality, which favor the hypothesis that A. drummondii served as paternal hybridization partner more frequently than A. holboellii.

Partial wave analysis of J/ ψ→ γ( K±KS0π∓)

BES data on J/ ψ→ γ( K ± K S 0 π ∓) are presented. There is a strong peak due to η(1440)/ ι, which is fitted with a Breit–Wigner amplitude with s-dependent widths for decays to K ∗K , κK, ηππ and ρρ; κ refers to the Kπ S-wave. At a K K ̄ π mass of ∼2040 MeV, there is a second peak with width ∼400 MeV; J P =0 − is preferred over 1 + and 2 − respectively by 5.2 and 6.8 standard deviations. It is a possible candidate for a 0 − s s ̄ g hybrid partner of π(1800).

Breeding of Polyploid Asparagus (Asparagus officinalis L.)

AbstractAsparagus is especially suitable for polyploidy breeding. The aim is to develop clone‐based hybrid varieties, in particular male 4 × varieties. For this purpose, genetically divergent diploid idiotypes*) are subjected to mitotic or meiotic polyploidization. In developing hybrid partners, special attention is paid to resistance in addition to yield and quality characters. Early diagnoses are applied in selection so as to shorten breeding times. Somaclonal variations may occur relatively often on in vitro propagation; these need to be eliminated. Triplex and quadruplex type males are suitable for paternal hybrid partners. Strong inbreeding depressions need to be avoided in their production.

Hybridoma Antibodies Produced against Bromelain Derived Cores of Influenza Virus

Splenic lymphocytes from BALB/c mice immunized with "cores" of influenza virus, obtained after bromelain cleavage of the surface glycoprotein, were fused with the P3-NS1/1-Ag-1 mouse cell line to yield hybridoma cultures. Among 20 stable cloned hybrid cells secreting monoclonal antibodies, one was specific for the nucleoprotein (NP), 11 were specific for the membrane (M) protein and eight were specific for the hemagglutinin (HA). These "cores" used as immunogen contained only the internal proteins of the influenza virus, namely the three polymerases, the NP and the M protein and no HA when examined by standard procedures of SDS-PAGE, electron microscopy and hemagglutination activity. It thus appeared that a small amount of contaminating antigens can sensitize a sufficient number of mouse B cells to be selected as hybrid partners. These antibodies were provisionally assigned as anti-carbohydrate attached to the HA.

Design of coated fuel particles for a hybrid fusion-fission system

Abstract Designs have been developed for coated ThO 2 fuel particles to be used in a hybrid fusion-fission system that could be operated without reprocessing. The fresh fertile fuel particle would first be cycled through the blanket of a fusion reactor to breed 233 U, which would then be ‘burned’ in a thermal fission reactor. The depleted fuel would then be refreshed in a second pass through the fusion reactor, and the process above repeated as many times as feasible. Designs of coated particles for up to three cycles through the hybrid system of reactors have been developed. The outer structural layer for these particles is made from vapor-deposited silicon carbide, because of its remarkable dimensional stability under fast neutron irradiation, and an inner layer of porous pyrocarbon is used to accommodate the buildup of gaseous reaction products inside the particle. The production of gaseous emission products from the interaction of high-energy fusion neutrons with coating materials and with the oxygen in the kernel contributes significantly to pressure vessel stresses in these coatings, whereas gaseous fission products alone dominate in conventional thermal reactors. The most stringent design for the three-cycle particle is identical in fuel loading to the reference fertile particle for an HTGR, which would constitute an ideal hybrid partner for the fusion reactor. Consideration is also given to coated-particle designs for the containment of the bred tritium used to fuel the D-T fusion reactor.

Mechanisms in early and late phases of tolerance induced by parabiosis in adult mice.

PARABIOSIS between CBAT6T6 inbred mice with the (CBA × C3H)F1 hybrid (=T6∼F1) (∼ is the symbol for parabiosis) gives considerable latitude in the study of the early phase of non-reactivity (specific immunological tolerance) induced by antigen because the cross circulation develops slowly between the partners1 and the CBA and (C3H inbred mouse strains from which the partners are derived are identical at the H-2 histocompatibility locus. This constitutes a weak antigenic combination and, in conjunction with the gradual appearance of the cross circulation, the resulting (one-way2) immunological reaction between the partners is mild, so that many survive for subsequent study of the mechanism by which tolerance of the foreign antigens present in the hybrid partner is induced in the parental partner.

IMMUNE MECHANISMS IN PARABIOSIS INTOXICATION.

When A strain mice are placed in parabiotic union with (A x C57Bl/1)F(1) hybrid partners, the parental strain partners are polycythemic and the hybrids anemic from the 5th through the 16th parabiosis days. All hybrids develop clinical intoxication between the 7th and the 12th days, and no pairs survive to 1 month. Long-term survival of parabiotic pairs can be achieved if lethally irradiated or specifically tolerant parental strain mice are united to hybrid partners. Production of tolerance by either of these methods results in elimination of anemia-polycythemia by the 12th parabiosis day and prevents intoxication in the hybrid partners. Preimmunization of the parental strain partners against the C57Bl/1 component of the hybrid leads to a considerable intensification of day 5 anemia-polycythemia. Intoxication develops in the hybrid partners between the 4th and the 6th days after union. It is concluded that anemia is primarily responsible for the syndrome of clinical intoxication. Early anemia-polycythemia on day 5 does not depend upon an immunological mechanism, but the late anemia-polycythemia appearing between days 12 and 16 is a function of the ability of the parental strain mouse to react immunologically against its hybrid partner. When neonatally thymectomized A strain mice are joined to hybrid partners, anemia-polycythemia is sustained through the 16th day and the hybrid partners develop clinical intoxication. On the other hand, when both partners are neonatally thymectomized, late anemia-polycythemia is considerably reduced, and the hybrid partners apparently do not develop clinical intoxication. It is concluded that normal hybrid mice are capable of reconstituting the immunological capacity of their thymectomized partners, whereas thymectomized hybrid mice do not have this restorative capacity. These findings are discussed in terms of their possible application to the problem of the induction of immunological tolerance in adult mice by the parabiosis procedure.

PARABIOSIS INTOXICATION IN INBRED RAT PAIRS AND ITS MODIFICATION BY X-IRRADIATION.

Parabiosis intoxication and its modification by x irradiation was investigated in inbred rat strains. Development of intoxication was followed by visual inspection and by taking microhematocrits from tail vein blood before parabiosis and on given days after pairing. Parabiotic groups consisted of Buffalo-Lewis, Lewis-LBF/sub 1/ (Lewis x Buffalo F/sub 1/), and Buffalo-LBF/sub 1/ strain combinations. Lewis rats were more prone to develop an early temporary anemia. Intoxication (anemia of one rat and polycythemia of the other) with fatality developed in most Buffalo-Lewis pairs. When LBF/sub 1/ animals were paired with either parent strain, anemia of intoxication was limited to the hybrid. In BuffaloLBF/sub 1/ combinations all hybrid partners became severely anemic and mortality was complete. Total-body x irradiation (500 r) of Buffalo rats immediately before parabiosis with LBF/sub 1/ partners eliminated intoxication and resultant mortality. In pairs in which the hybrid partner was irradiated, anemia-polycythemia was initiated more rapidly and mortality was high. Irradiation of both partners reduced hematocrit changes but did not prolong survival. Intoxication was interpreted as an immune response of the parent against the hybrid; elimination of the syndrome by irradiation of the parent supports this hypothesis.

Tolerance to Skin Homografts of Adult Mice after Parabiosis

SKIN grafts between mice of different inbred strains are invariably rejected within a few weeks. Hybrid (F 1) tissues transplanted to mice of either parental strain are also consistently rejected at the same rate if the parents differ at the H-2 histocompatibility locus1. This uniform incompatibility was not apparent when such hybrids were united in parabiosis with parental strain inbred mice. The most frequent result of such unions was the death of only the hybrid partner, usually within a month. Hybrid death in this period was characteristically preceded by a severe wasting disease, sometimes described as ‘parabiotic intoxication’2.