Homologous recombination in Agrobacterium: potential implications for the genomic species concept in bacteria

According to current taxonomical rules, a bona fide bacterialspecies is a genomic species characterized by the genomic similarityof its members. It has been proposed that the genomic cohesionof such clusters may be related to sexual isolation, which limitsgene flow between too divergent bacteria. Homologous recombinationis one of the most studied mechanisms responsible for this geneticisolation. Previous studies on several bacterial models showedthat recombination frequencies decreased exponentially withincreasing DNA sequence divergence. In the present study, weinvestigated this relationship in the Agrobacterium tumefaciensspecies complex, which allowed us to focus on sequence divergencein the vicinity of the genetic boundaries of genomic species.We observed that the sensitivity of the recombination frequencyto DNA divergence fitted a log-linear function until approximately10% sequence divergence. The results clearly revealed that therewas no sharp drop in recombination frequencies at the pointwhere the sequence divergence distribution showed a "gap" delineatinggenomic species. The ratio of the recombination frequency inhomogamic conditions relative to this frequency in heterogamicconditions, that is, sexual isolation, was found to decreasefrom 8 between the most distant strains within a species to9 between the most closely related species, for respective increasesfrom 4.3% to 6.4% mismatches in the marker gene chvA. This meansthat there was only a 1.13-fold decrease in recombination frequenciesfor recombination events at both edges of the species border.Hence, from the findings of this investigation, we concludethat—at least in this taxon—sexual isolation basedon homologous recombination is likely not high enough to stronglyhamper gene flow between species as compared with gene flowbetween distantly related members of the same species. The 70%relative binding ratio cutoff used to define bacterial speciesis likely correlated to only minor declines in homologous recombinationfrequencies. Consequently, the sequence diversity, as a mechanisticfactor for the efficiency of recombination (as assayed in thelaboratory), appears to play little role in the genetic cohesionof bacterial species, and thus, the genomic species definitionfor prokaryotes is definitively not reconcilable with the biologicalspecies concept for eukaryotes.     

Development of the amentiferous concept

The amentiferous concept developed in pre-Linnaean times, and early botanists clearly recognized the topical similarities among plants bearing aments. Among the amentiferous plants placed side by side in early times were many that would not be so situated today—e.g., gymnosperms intermixed with dicotyledons. By the time of Linnaeus, only dicotyledons were included among the ament-bearing groups. J. G. Gmelin was first to recognize ament-bearing plants (including some gymnosperms) under a single category, “Amentaceae.” Linnaeus, A. L. de Jussieu, W. J. Hooker, Lindley, and Eichler, at one time or another, placed these plants in a separate amentaceous category, but never under the term “Amentiferae.” The name was never used by Engler although he did place the ament-bearing plants among the first families of his Archichlamydeae. The category “Amentiferae” appears to have entered the literature in British publications and through British/English translations from the German.     

Charge-exchange recombination spectroscopy of the plasma ion temperature at the T-10 tokamak

Charge-exchange recombination spectroscopy (CXRS) based on a diagnostic neutral beam has been developed at the T-10 tokamak. The diagnostics allows one to measure the ion temperature profile in the cross section of the plasma column. In T-10 experiments, the measurement technique was adjusted and the elements of the CXRS diagnostics for ITER were tested. The used spectroscopic equipment makes it possible to reliably determine the ion temperature from the Doppler broadening of impurity lines (helium, carbon), as well as of the spectral lines of the working gas. The profiles of the plasma ion temperature in deuterium and helium discharges were measured at different plasma currents and densities, including with the use of active Doppler measurements of lines of different elements. The validity and reliability of ion temperature measurements performed by means of the developed CXRS diagnostics are analyzed.     

A concept of eliminating nonhomologous recombination for scalable and safe AAV vector generation for human gene therapy

Scalable and efficient production of high-quality recombinant adeno-associated virus (rAAV) for gene therapy remains a challenge despite recent clinical successes. We developed a new strategy for scalable and efficient rAAV production by sequestering the AAV helper genes and the rAAV vector DNA in two different subcellular compartments, made possible by using cytoplasmic vaccinia virus as a carrier for the AAV helper genes. For the first time, the contamination of replication-competent AAV particles (rcAAV) can be completely eliminated in theory by avoiding ubiquitous nonhomologous recombination. Vector DNA can be integrated into the host genomes or delivered by a nuclear targeting vector such as adenovirus. In suspension HeLa cells, the achieved vector yield per cell is similar to that from traditional triple-plasmid transfection method. The rcAAV contamination was undetectable at the limit of our assay. Furthermore, this new concept can be used not only for production of rAAV, but also for other DNA vectors.     

Development of the concept of immunologic specificity: IV

We have attempted, in this series of essays on the development of the concept of immunologic specificity, to trace the history of one of the most central ideas in immunology (and indeed in biology in general). The result must be viewed as preliminary and incomplete and as an invitation to others to add, to amend, and even to disprove. Nevertheless, several interesting conclusions may be drawn, which reveal much about the workings of immunology in particular, and perhaps science in general. First, the roots of any important scientific concept (such as that of immunologic specificity) do not grow in isolation; they draw nourishment from many other disciplines. Similarly, the growth of an important concept within a given discipline will have far-reaching implications and fruits for other fields of science. Second, we may note a marked change in the manner in which immunology is currently practiced, compared with that of the end of the last century. The quantum leaps forward in funding, in numbers of scientists, and in masses of crucial data have not been without a certain cost--the substantial reduction in elegant personal style that characterized so many of our scientific forebears, and that makes so pleasant the reading of their reports. Finally, we see again and again how much his philosophical bases and disciplinary upbringing determine a scientist's approach, the questions that he asks, and the type of answers he will accept. Throughout much of immunology's history, as Jerne put it so well (44), cis- and trans-immunologists hardly spoke to one another. Or rather, a cis-immunologist sometimes spoke to a trans-immunologist, but the latter rarely answered! Fortunately, one of the attributes of scientific progress is a merging of these disparate languages, and eventual mutual comprehension.     

Development of an immunosensor for the detection of vitellogenin using impedance spectroscopy

A simple and direct immunosensor for the determination of carp (Carassius auratus) vitellogenin (Vtg), a female-specific protein, has been proposed based on an antibody-captured conducting polymer-coated electrode. The monoclonal antibody specific to carp (C. auratus) Vtg was immobilized by covalent coupling to the carboxylic acid group on the polymer. The antibody immobilization and antibody-antigen interaction have been demonstrated by means of quartz crystal microbalance and impedance spectroscopic techniques. The impedance change occurred at the sensor surface due to the specific immuno-interaction was utilized to determine Vtg. The sensor showed high selectivity and sensitive response to Vtg in a buffered medium without redox probe. Vtg was determined in the linear range from 1.0 to 8.0 microg/l with the standard deviation of +/-0.13 (n =3) and the detection limit was determined to be 0.42 microg/l. This method was applied to the determination of Vtg in real male and female carp (C. auratus) serum samples.     

Development of a 1H NMR structural-reporter-group concept for the primary structural characterisation of alpha-D-glucans

An NMR study of proton chemical shift patterns of known linear alpha-D-glucopyranose di- and trisaccharide structures was carried out. Chemical shift patterns for (alpha1-->2)-, (alpha1-->3)-, (alpha1-->4)- and (alpha1-->6)-linked D-glucose residues were analysed and compared to literature data. Using these data, a 1H NMR structural-reporter-group concept was formulated to function as a tool in the structural analysis of alpha-D-glucans.     

Formation of a charge-exchange target for fast ions in the plasma of large-scale toroidal devices under NBI conditions

High-energy (E>0.2 MeV) charge-exchange diagnostics allow the determination of the distribution function of fast atoms produced via the neutralization of hydrogen isotope ions by target hydrogen-like impurity ions. To derive the distribution function from the experimental data requires knowledge of the composition and spatial distribution of the target ions in a tokamak plasma. A charge-exchange target forms as a result of the interaction between the main impurity nuclei and the heating neutral beams. In different devices, the heating beams are arranged in different ways with respect to the diagnostics; hence, in order to accurately estimate the contribution of the secondary ions to the detected signal, it is necessary to calculate their trajectories for every particular case. A model is proposed that takes into account elementary processes resulting in the ionization equilibrium of the ions of different impurities with allowance for ion motion in a specific tokamak configuration. As an example, the model is applied to the plasma of the JT-60U tokamak. Mechanisms for the formation of charge-exchange atomic flows in various energy ranges are considered. The relative contributions of different heating injectors to the charge-exchange flow are estimated. Based on the calculated results, a method is proposed for local measurements of the ion distribution function with the help of a stationary analyzer.     

A case for the extreme antiquity of recombination

Recombination is usually assumed to be a mode of reproduction that evolved long after asexual reproduction in response to specific genetic and environmental circumstances. Here the argument is made that recombination was an evolutionary development as ancient as the origins of life. To support this proposition four lines of evidence are given, in particular, the need for primordial genomes to acquire substantial length and to escape from Mullers Ratchet.     

Development of a Climate Choice meal concept for restaurants based on carbon footprinting

Purpose

Significant reductions in greenhouse gas emissions from food production and consumption can be made at the level of individual diet. Together with the food and beverage sector, consumers could play a significant role by making informed choices that benefit the environment and their own health. Communicating information on carbon footprints to consumers is challenging and should be made very simple, yet reliable. This sector is showing interest in using eco-design tools to decrease climate change impacts of their meals.

Methods

A long-term concept for communicating information on carbon footprints associated with meals was developed in Finland. The criteria for a Climate Choice meal were created through stakeholder dialogue, and three restaurant operators piloted the concept in 25 restaurants. In addition to climate change impacts, possibilities to include other sustainability criteria were reviewed. The concept was based on simplified carbon footprinting of raw material production and processing of ingredients for 105 commonly selected lunches. The carbon footprint calculations allowed the development of the Climate Choice meal concept, its criteria, and piloting the concept. Based on experiences from restaurants and consumers from the pilot phase, final criteria were developed.

Results and discussion

The Climate Choice meal concept was created using two alternative climate criteria: one for immediate implementation and another for future implementation, in cases where carbon footprinting is feasible for restaurants. The criteria for immediate implementation include a list of mainly plant-based ingredients with low carbon footprint. Regarding future criteria, it should be made easy enough for restaurants to estimate the carbon footprints of their meals, allowing labeling of meals when their carbon footprints are at least 25 % smaller than for an average meal. In addition to the two climate criteria, Climate Choice meals need to follow Finnish public catering nutritional recommendations, taking into account that fish species on the Red List of WWF’s Finnish seafood guide are prohibited.

Conclusions

To promote climate-friendly eating, a long-term concept rather than a short-term campaign is needed. There is interest among consumers and restaurants for information on food carbon footprints and sustainability. Lunch is regarded as a good opportunity for consumers to learn about climate-friendly eating. The main challenges are to produce sufficiently reliable background data and to raise consumer and the food and beverage sector interest and understanding of carbon footprints associated with food.

     

Quantitation of membrane receptor distributions by image correlation spectroscopy: concept and application.

Measurement of receptor distributions on cell surfaces is one important aspect of understanding the mechanism whereby receptors function. In recent years, scanning fluorescence correlation spectroscopy has emerged as an excellent tool for making quantitative measurements of cluster sizes and densities. However, the measurements are slow and usually require fixed preparations. Moreover, while the precision is good, the accuracy is limited by the relatively small amount of information in each measurement, such that many are required. Here we present a novel extension of the scanning correlation spectroscopy that solves a number of the present problems. The new technique, which we call image correlation spectroscopy, is based on quantitative analysis of confocal scanning laser microscopy images. Since these can be generated in a matter of a second or so, the measurements become more rapid. The image is collected over a large cell area so that more sampling is done, improving the accuracy. The sacrifice is a lower resolution in the sampling, which leads to a lower precision. This compromise of precision in favor of speed and accuracy still provides an enormous advantage for image correlation spectroscopy over scanning correlation spectroscopy. The present work demonstrates the underlying theory, showing how the principles can be applied to measurements on standard fluorescent beads and changes in distribution of receptors for platelet-derived growth factor on human foreskin fibroblasts.     

Formation of charge-exchange ion flows near the exit from the stationary plasma thruster acceleration channel

Ion currents onto the exit plane of the acceleration channel of a stationary plasma thruster model were measured using electrostatic probes the collecting surfaces of which could be oriented either upstream or downstream with respect to the thruster plume. Using the results of measurements, the so-called “back” flows of charge-exchange ions onto the exit plane are estimated. It is shown that the back ion flows are the most intense in the close vicinity of the thruster, but do not exceed 0.6% of the total ion flow from the thruster. The formation of steady-state ion flows near the exit from the acceleration channel of a stationary plasma thruster is simulated numerically by using a three-dimensional kinetic model that describes the dynamics of ions and neutral atoms exhausting from the acceleration channel and produced in the thruster plume and takes into account resonance charge exchange of ions with neutral atoms. The distribution of the back ion current density in the exit plane is determined. The effect of the flow rate of the working gas through the cathode on the distributions of the neutral atom density and charge-exchange ion flows is demonstrated. The obtained results can be used to analyze the effect of the thruster plume on the charge state of the surfaces located in the vicinity of the thruster.     

Ultrasonic wave spectroscopy study of sugar oligomers and polysaccharides in aqueous solutions: the hydration length concept

The determination of apparent persistence length and radius of gyration of maltodextrins in water is achievable through high-resolution ultrasonic spectroscopy measurements. Classical hydration number for those carbohydrates is characteristic of an apparent persistence degree of polymerisation of the polymer. A force-field based molecular modeling of a 10DP malto-oligomer allows measurement of the corresponding length for the lowest energetic conformation in solution. A good agreement between the apparent radii of gyration determined by this technique and the freely rotating polymer chain model is found with radii of gyration calculated from the intrinsic viscosity.     

Development of a viral insecticide: concept to commercialization

About 18 months ago, on December 8, 1970, the Food and Drug Administration officially granted the Heliothis nucleo polyhedrosis virus (NPV) the status of temporary exemption from requirement of a tolerance. This NPV, registered for use against Heliothis on cotton, has also been extensively tested on Heliothis species attacking corn, sorghum, tobacco and truck crops. This was the first time an insect virus had been officially registered by a federal agency for use as an insecticide. More recently Spain has registered this virus, and other governments also are in the process of registering the virus.The historical and technical development of this virus from concept to commercialization is the subject of this presentation. The virus, first isolated from diseased Heliothis attacking cotton in the Rio Grande Valley in Texas, was successfully developed through laboratory, pilot-plant, and commercial phases. At each phase the virus was evaluated on the basis of: (1) its safety to man, animals, and plants; (2) its production feasibility and costs; and (3) its effectiveness against the specific target pest. Currently, two companies (International Minerals and Chemical Corporation, Libertyville, Ill., and Nutrilite Products, Inc., Buena Vista, Calif.) are producing the Heliothis NPV under their trade names Viron/H and Biotrol VHZ, respectively, for experimental programs and for limited sales.     

A de novo recombination in the ABO blood group gene and evidence for the occurrence of recombination products

We have encountered a paternity case where exclusion of the putative father was only observed in the ABO blood group (mother, B; child, A1; putative father, O), among the many polymorphic markers tested, including DNA fingerprints and microsatellite markers. Cloning a part of the ABO gene, PCR-amplified from the trio’s genomes, followed by sequencing the cloned fragments, showed that one allele of the child had a hybrid nature, comprising exon 6 of the B allele and exon 7 of the O1 allele. Based on the evidence that exon 7 is crucial for the sugar-nucleotide specificity of A1 and B transferases and that the O1 allele is only specified by the 261G deletion in exon 6 of the consensus sequence of the A1 allele, we concluded that the hybrid allele encodes a transferase with A1 specificity, resulting, presumably, from de novo recombination between the B and O1 alleles of the mother during meiosis. Screening of random populations demonstrated the occurrence of four other hybrid alleles. Sequencing of intron VI from the five hybrid alleles showed that the junctions of the hybrid alleles were located within intron VI, the intron VI-exon 7 boundaries, or exon 7. Recombinational events seem to be partly involved in the genesis of sequence diversities of the ABO gene. Received: 25 October 1996     

Analysis of European mtDNAs for recombination

The standard paradigm postulates that the human mitochondrial genome (mtDNA) is strictly maternally inherited and that, consequently, mtDNA lineages are clonal. As a result of mtDNA clonality, phylogenetic and population genetic analyses should therefore be free of the complexities imposed by biparental recombination. The use of mtDNA in analyses of human molecular evolution is contingent, in fact, on clonality, which is also a condition that is critical both for forensic studies and for understanding the transmission of pathogenic mtDNA mutations within families. This paradigm, however, has been challenged recently by Eyre-Walker and colleagues. Using two different tests, they have concluded that recombination has contributed to the distribution of mtDNA polymorphisms within the human population. We have assembled a database that comprises the complete sequences of 64 European and 2 African mtDNAs. When this set of sequences was analyzed using any of three measures of linkage disequilibrium, one of the tests of Eyre-Walker and colleagues, there was no evidence for mtDNA recombination. When their test for excess homoplasies was applied to our set of sequences, only a slight excess of homoplasies was observed. We discuss possible reasons that our results differ from those of Eyre-Walker and colleagues. When we take the various results together, our conclusion is that mtDNA recombination has not been sufficiently frequent during human evolution to overturn the standard paradigm.     

Development of an indole-based chemically cleavable linker concept for immobilizing bait compounds for protein pull-down experiments

A novel linker chemistry based on a malondialdehyde-indole condensation reaction has been developed for the affinity-independent elution of targeted protein pull-downs. Previously developed in our lab for the tagging of tryptophan residues on proteins or peptides, the concept was extended for the design of a chemically cleavable linker system. Target molecules for interaction studies are immobilized on a solid support including the linker scaffold, and a typical pull-down experiment is carried out. After purification, the linker is cleaved by incubation with 50 mM pyrrolidine. A specific tyrosine kinase inhibitor, bosutinib, was coupled to agarose and acrylamide beads, respectively, via the new linker system, and a protein pull-down experiment of putative interaction partners from a K562 whole cell lysate was performed. The system was found to be compatible with targeted protein pull-downs; during the cleavage step, no protein hydrolysis or any degradation of amino acid side-chains was apparent. From the pull-down experiment, key targets of bosutinib such as the tyrosine kinase, Btk, were identified by liquid chromatography-tandem mass spectrometry.