Detection of transgenic and endogenous plant DNA fragments in the blood, tissues, and digesta of broilers

The aim was to determine the fate of transgenic and endogenous plant DNA fragments in the blood, tissues, and digesta of broilers. Male broiler chicks (n = 24) were allocated at 1 day old to each of four treatment diets designated T1-T4. T1 and T2 contained the near isogenic nongenetically modified (GM) maize grain, whereas T3 and T4 contained GM maize grain [cry1a(b) gene]; T1 and T3 also contained the near isogenic non-GM soybean meal, whereas T2 and T4 contained GM soybean meal (cp4epsps gene). Four days prior to slaughter at 39-42 days old, 50% of the broilers on T2-T4 had the source(s) of GM ingredients replaced by their non-GM counterparts. Detection of specific DNA sequences in feed, tissue, and digesta samples was completed by polymerase chain reaction analysis. Seven primer pairs were used to amplify fragments (similar to 200 bp) from single copy genes (maize high mobility protein, soya lectin, and transgenes in the GM feeds) and multicopy genes (poultry mitochondrial cytochrome b, maize, and soya rubisco). There was no effect of treatment on the measured growth performance parameters. Except for a single detection of lectin (nontransgenic single copy gene; unsubstantiated) in the extracted DNA from one bursa tissue sample, there was no positive detection of any endogenous or transgenic single copy genes in either blood or tissue DNA samples. However, the multicopy rubisco gene was detected in a proportion of samples from all tissue types (23% of total across all tissues studied) and in low numbers in blood. Feed-derived DNA was found to survive complete degradation up to the large intestine. Transgenic DNA was detected in gizzard digesta but not in intestinal digesta 96 h after the last feeding of treatment diets containing a source of GM maize and/or soybean meal.

Detection of transgenic DNA and protein, in rumen fluid, duodenal digesta, milk, blood and faeces of lactating dairy cows

The objective was to determine the presence or absence of transgenic and endogenous plant DNA in ruminal fluid, duodenal digesta, milk, blood, and feces, and if found, to determine fragment size. Six multiparous lactating Holstein cows fitted with ruminal and duodenal cannulas received a total mixed ration. There were two treatments (T). In T1, the concentrate contained genetically modified (GM) soybean meal (cp4epsps gene) and GM corn grain (cry1a[b] gene), whereas T2 contained the near isogenic non-GM counterparts. Polymerase chain reaction analysis was used to determine the presence or absence of DNA sequences. Primers were selected to amplify small fragments from single-copy genes (soy lectin and corn high-mobility protein and cp4epsps and cry1a[b] genes from the GM crops) and multicopy genes (bovine mitochondrial cytochrome b and rubisco). Single-copy genes were only detected in the solid phase of rumen and duodenal digesta. In contrast, fragments of the rubisco gene were detected in the majority of samples analyzed in both the liquid and solid phases of ruminal and duodenal digesta, milk, and feces, but rarely in blood. The size of the rubisco gene fragments detected decreased from 1176 bp in ruminal and duodenal digesta to 351 bp in fecal samples.

Detection of transgenic and endogenous plant DNA in rumen fluid, duodenal digesta, milk, blood, and feces of lactating dairy cows

The objective was to determine the presence or absence of transgenic and endogenous plant DNA in ruminal fluid, duodenal digesta, milk, blood, and feces, and if found, to determine fragment size. Six multiparous lactating Holstein cows fitted with ruminal and duodenal cannulas received a total mixed ration. There were two treatments (T). In T1, the concentrate contained genetically modified (GM) soybean meal (cp4epsps gene) and GM corn grain (cry1a[b] gene), whereas T2 contained the near isogenic non-GM counterparts. Polymerase chain reaction analysis was used to determine the presence or absence of DNA sequences. Primers were selected to amplify small fragments from single-copy genes (soy lectin and corn high-mobility protein and cp4epsps and cry1a[b] genes from the GM crops) and multicopy genes (bovine mitochondrial cytochrome b and rubisco). Single-copy genes were only detected in the solid phase of rumen and duodenal digesta. In contrast, fragments of the rubisco gene were detected in the majority of samples analyzed in both the liquid and solid phases of ruminal and duodenal digesta, milk, and feces, but rarely in blood. The size of the rubisco gene fragments detected decreased from 1176 bp in ruminal and duodenal digesta to 351 bp in fecal samples.

Dynamics of QoI sensitivity in Mycosphaerella fijiensis in Costa Rica during 2000 to 2003

From 1997 onward, the strobilurin fungicide azoxystrobin was widely used in the main banana-production zone in Costa Rica against Mycosphaerella fijiensis var. difformis causing black Sigatoka of banana. By 2000, isolates of M. fijiensis with resistance to the quinolene oxidase inhibitor fungicides were common on some farms in the area. The cause was a single point mutation from glycine to alanine in the fungal target protein, cytochrome b gene. An amplification refractory mutation system Scorpion quantitative polymerase chain reaction assay was developed and used to determine the frequency of G 143A allele in samples of M. fijiensis. Two hierarchical surveys of spatial variability, in 2001 and 2002,found no significant variation in frequency on spatial scales <10 in. This allowed the frequency of G143A alleles on a farm to be estimated efficiently by averaging single samples taken at two fixed locations. The frequency of G 143A allele in bulk samples from I I farms throughout Costa Rica was determined at 2-month intervals. There was no direct relationship between the number of spray applications and the frequency of G143A on individual farms. Instead, the frequency converged toward regional averages, presumably due to the large-scale mixing of ascospores dispersed by wind. Using trap plants in an area remote from the main producing area, immigration of resistant ascospores was detected as far as 6 km away both with and against the prevailing wind.

Use of a novel Hill-climbing genetic algorithm in protein folding simulations

We have developed a novel Hill-climbing genetic algorithm (GA) for simulation of protein folding. The program (written in C) builds a set of Cartesian points to represent an unfolded polypeptide's backbone. The dihedral angles determining the chain's configuration are stored in an array of chromosome structures that is copied and then mutated. The fitness of the mutated chain's configuration is determined by its radius of gyration. A four-helix bundle was used to optimise simulation conditions, and the program was compared with other, larger, genetic algorithms on a variety of structures. The program ran 50% faster than other GA programs. Overall, tests on 100 non-redundant structures gave comparable results to other genetic algorithms, with the Hill-climbing program running from between 20 and 50% faster. Examples including crambin, cytochrome c, cytochrome B and hemerythrin gave good secondary structure fits with overall alpha carbon atom rms deviations of between 5 and 5.6 Angstrom with an optimised hydrophobic term in the fitness function. (C) 2003 Elsevier Ltd. All rights reserved.

Global iron-dependent gene regulation in Escherichia coli - A new mechanism for iron homeostasis

Organisms generally respond to iron deficiency by increasing their capacity to take up iron and by consuming intracellular iron stores. Escherichia coli, in which iron metabolism is particularly well understood, contains at least 7 iron-acquisition systems encoded by 35 iron-repressed genes. This Fe-dependent repression is mediated by a transcriptional repressor, Fur ( ferric uptake regulation), which also controls genes involved in other processes such as iron storage, the Tricarboxylic Acid Cycle, pathogenicity, and redox-stress resistance. Our macroarray-based global analysis of iron- and Fur-dependent gene expression in E. coli has revealed several novel Fur-repressed genes likely to specify at least three additional iron- transport pathways. Interestingly, a large group of energy metabolism genes was found to be iron and Fur induced. Many of these genes encode iron- rich respiratory complexes. This iron- and Fur-dependent regulation appears to represent a novel iron-homeostatic mechanism whereby the synthesis of many iron- containing proteins is repressed under iron- restricted conditions. This mechanism thus accounts for the low iron contents of fur mutants and explains how E. coli can modulate its iron requirements. Analysis of Fe-55-labeled E. coli proteins revealed a marked decrease in iron- protein composition for the fur mutant, and visible and EPR spectroscopy showed major reductions in cytochrome b and d levels, and in iron- sulfur cluster contents for the chelator-treated wild-type and/or fur mutant, correlating well with the array and quantitative RT-PCR data. In combination, the results provide compelling evidence for the regulation of intracellular iron consumption by the Fe2+-Fur complex.

Wolbachia infection and dramatic intraspecific mitochondrial dna divergence in a fig wasp

Mitochondria and Wolbachia are maternally inherited genomes that exhibit strong linkage disequilibrium in many organisms. We surveyed Wolbachia infections in 187 specimens of the fig wasp species, Ceratosolen solmsi, and found an infection prevalence of 89.3%. DNA sequencing of 20 individuals each from Wolbachia-infected and -uninfected subpopulations revealed extreme mtDNA divergence (up to 9.2% and 15.3% in CO1 and cytochrome b, respectively) between infected and uninfected wasps. Further, mtDNA diversity was significantly reduced within the infected group. Our sequencing of a large part of the mitochondrial genome from both Wolbachia-infected and -uninfected individuals revealed that high sequence divergence is common throughout the mitochondrial genome. These patterns suggest a partial selective sweep of mitochondria subsequent to the introduction of Wolbachia into C. solsmi, by hybrid introgression from a related species.

Temporal genetic variation of the red fox, Vulpes vulpes, across western Europe and the British Isles

Quaternary climatic fluctuations have had profound effects on the phylogeographic structure of many species. Classically, species were thought to have become isolated in peninsular refugia, but there is limited evidence that large, non-polar species survived outside traditional refugial areas. We examined the phylogeographic structure of the red fox (Vulpes vulpes), a species that shows high ecological adaptability in the western Palaearctic region. We compared mitochondrial DNA sequences (cytochrome b and control region) from 399 modern and 31 ancient individuals from across Europe. Our objective was to test whether red foxes colonised the British Isles from mainland Europe in the late Pleistocene, or whether there is evidence that they persisted in the region through the Last Glacial Maximum. We found red foxes to show a high degree of phylogeographic structuring across Europe and, consistent with palaeontological and ancient DNA evidence, confirmed via phylogenetic indicators that red foxes were persistent in areas outside peninsular refugia during the last ice age. Bayesian analyses and tests of neutrality indicated population expansion. We conclude that there is evidence that red foxes from the British Isles derived from central European populations that became isolated after the closure of the landbridge with Europe.

Erratum to: “Photoproduction of heavy quarks in next-to-leading order QCD with longitudinally polarized initial states” [Nucl. Phys. B 540 (1999) 345]

There are three trivial misprints in our paper.

Evidence of heavy metal movement down the profile of a heavily-sludged soil

Two sites in central England where sewage sludge has been deposited for decades were studied to measure the heavy metal distribution in the soil profiles. The first site (S 1) was a field receiving heavy loads sludge from a nearby wastewater treatment plant, and the second (S2) was a farm applying 'normal' sludge rates of 8 t ha(-1) y(-1) of the same sludge. Soil samples were also taken by a near-by untreated control site. In S I the movement of heavy metals was significant even down to 80 cm depth compared to the control. In S2, the concentrations of lead (Pb) and zinc (Zn) and the organic matter content were higher than the control down to 20 cm, while nickel (Ni) moved significantly down to 80 cm. This underlies. the possibility that the metals bound onto organic surfaces moved along with organic matter down to that depth. The movement of metals in S2 points out the potential risks of applying sewage sludge for a long time.

Influence of dissolved organic matter on the solubility of heavy metals in sewage-sludge-amended soils

Sewage-sludge-amended soils generally contain elevated levels of organic matter and heavy metals compared to control soils. Because organic matter is known to complex with heavy metals, the solubility behavior of the organic matter in such soils may exert a significant influence on the solubility of the metals. Little is known about such a process. Using batch experiments in which the solubility of organic matter in a heavily sludge-amended soil was artificially manipulated, we show that the solubilities of the heavy metals copper (Cu), nickel (Ni), and lead (Pb) show a strong positive relationship to the solubility of organic matter, particularly at high pH. The results suggest that under field conditions, spatiotemporal variations in the solid-solution partitioning of organic matter may have a bearing on the environmental significance (mobility and bioavailability) of these heavy metals.

An inventory of heavy metals inputs to agricultural soils in England and Wales

An inventory of heavy metal inputs (Zn, Cu, Ni, Pb, Cd, Cr, As and Hg) to agricultural soils in England and Wales in 2000 is presented, accounting for major sources including atmospheric deposition, sewage sludge, livestock manures, inorganic fertilisers and lime, agrochemicals, irrigation water, industrial by-product 'wastes' and composts. Across the whole agricultural land area, atmospheric deposition was the main source of most metals, ranging from 25 to 85% of total inputs. Livestock manures and sewage sludge were also important sources, responsible for an estimated 37-40 and 8-17% of total Zn and Cu inputs, respectively. However, at the individual field scale sewage sludge, livestock manures and industrial wastes could be the major source of many metals where these materials are applied. This work will assist in developing strategies for reducing heavy metal inputs to agricultural land and effectively targeting policies to protect soils from long-term heavy metal accumulation. (C) 2003 Elsevier Science B.V. All rights reserved.

Photoproduction of heavy quarks in next-to-leading order QCD with longitudinally polarized initial states

We present all relevant details of our calculation of the complete next-to-leading order O(αS2α) QCD corrections to heavy flavor photoproduction with longitudinally polarized point-like photons and hadrons. In particular we provide analytical results for the virtual plus soft gluon cross section. We carefully address the relevance of remaining theoretical uncertainties by varying, for instance, the factorization and renormalization scales independently. Such studies are of importance for a meaningful first direct determination of the polarized gluon density Δg from the total charm production spin asymmetry by the upcoming COMPASS experiment. It is shown that the scale uncertainty is considerably reduced in next-to-leading order, but the dependence on the charm quark mass is sizable at fixed target energies. Finally, we study several differential single-inclusive heavy quark distributions and, for the polarized HERA option, the total bottom spin asymmetry.

Towards polarized hadroproduction of heavy quarks

Though leading order (LO) predictions for the hadroproduction of heavy quarks at RHIC look promising, next-to-leading order (NLO) corrections must be considered. First partonic NLO results are reported here.

Next-to-leading order QCD corrections to the polarized photoproduction of heavy flavors

We present a calculation of the next-to-leading order ... QCD corrections to heavy flavor photoproduction with longitudinally polarized beams. We apply our results to study the longitudinal spin asymmetry for the total charm quark production cross section which will be utilized by the forthcoming COMPASS experiment at CERN to obtain first direct information on the polarized gluon density Δg. We also briefly discuss the main theoretical uncertainties inherent in this calculation. In particular we demonstrate that the factorization scale dependence is considerably reduced in next-to-leading order.