Molecular biology. Targeting intron insertion into DNA

In work reported on page 452, researchers have found a way to coax certain introns, bits of genetic debris that litter the DNA and interrupt the coding sequences of many genes, to hop into the exact sequences where the researchers want them. The method could enhance all sorts of genetic manipulations, from studying basic gene function to combating viral infections to delivering genes for gene therapy.     

Structure of the quaternary complex of interleukin-2 with its alpha, beta, and gammac receptors

Interleukin-2 (IL-2) is an immunoregulatory cytokine that acts through a quaternary receptor signaling complex containing alpha (IL-2Ralpha), beta (IL-2Rbeta), and common gamma chain (gc) receptors. In the structure of the quaternary ectodomain complex as visualized at a resolution of 2.3 angstroms, the binding of IL-2Ralpha to IL-2 stabilizes a secondary binding site for presentation to IL-2Rbeta. gammac is then recruited to the composite surface formed by the IL-2/IL-2Rbeta complex. Consistent with its role as a shared receptor for IL-4, IL-7, IL-9, IL-15, and IL-21, gammac forms degenerate contacts with IL-2. The structure of gammac provides a rationale for loss-of-function mutations found in patients with X-linked severe combined immunodeficiency diseases (X-SCID). This complex structure provides a framework for other gammac-dependent cytokine-receptor interactions and for the engineering of improved IL-2 therapeutics.     

Designed protein-protein association

The analysis of natural contact interfaces between protein subunits and between proteins has disclosed some general rules governing their association. We have applied these rules to produce a number of novel assemblies, demonstrating that a given protein can be engineered to form contacts at various points of its surface. Symmetry plays an important role because it defines the multiplicity of a designed contact and therefore the number of required mutations. Some of the proteins needed only a single side-chain alteration in order to associate to a higher-order complex. The mobility of the buried side chains has to be taken into account. Four assemblies have been structurally elucidated. Comparisons between the designed contacts and the results will provide useful guidelines for the development of future architectures.     

Laser-induced ferroelectric structural order in an organic charge-transfer crystal

We report the direct observation by x-ray diffraction of a photoinduced paraelectric-to-ferroelectric structural phase transition using monochromatic 100-picosecond synchrotron pulses. It occurs in tetrathiafulvalene-p-chloranil, a charge-transfer molecular material in which electronic and structural changes are strongly coupled. An optical 300-femtosecond laser pulse switches the material from a neutral to an ionic state on a 500-picosecond time scale and, by virtue of intrinsic cooperativity, generates self-organized long-range structural order. The x-ray data indicate a macroscopic ferroelectric reorganization after the laser irradiation. Refinement of the structures before and after laser irradiation indicates structural changes at the molecular level.     

Genetic variability of six indigenous goat breeds using major histocompatibility complex-associated microsatellite markers

The present study aimed at analyzing the genetic variability of indigenous goat breeds (Capra hircus) using the MHC-associated microsatellite markers BF1, BM1818, BM1258, DYMS1, and SMHCC1. The following breeds were included: Chinese Xuhuai, Indian Changthangi and Pashmina, Kenyan Small East African (SEA) and Galla, and Albanian Vendi. To examine genetic variability, the levels of heterozigosity, degrees of inbreeding, and genetic differences among the breeds were analyzed. The mean number of alleles ranged from nine in the Galla to 14.5 in the Vendi breed. The mean observed heterozygosity and mean expected heterozygosity varied from 0.483 in the Vendi to 0.577 in the Galla breed, and from 0.767 in the SEA to 0.879 in the Vendi breed, respectively. Significant loss of heterozygosity (p < 0.01) indicated that these loci were not in Hardy-Weinberg equilibrium. The mean F_IS values ranged from 0.3299 in the SEA to 0.4605 in the Vendi breed with a mean value of 0.3623 in all breeds (p < 0.001). Analysis of molecular variance indicated that 7.14% and 4.74% genetic variation existed among the different breeds and geographic groups, whereas 92.86% and 95.26% existed in the breeds and the geographic groups, respectively (p < 0.001). The microsatellite marker analysis disclosed a high degree of genetic polymorphism. Loss of heterozygosity could be due to genetic drift and endogamy. The genetic variation among populations and geographic groups does not indicate a correlation of genetic differences with geographic distance.     

EMS and UV irradiation induce unstable resistance against CAA fungicides in <Emphasis Type="Italic">Bremia lactucae</Emphasis>

Wild type (WT) field isolates of Bremia lactucae failed to germinate in vitro or infect lettuce leaves in the presence of CAA (carboxylic acid amide) fungicides. Minimal inhibitory concentrations (MIC) for mandipropamid, dimethomorph, benthiavalicarb and iprovalicarb were 0.005, 0.5, 0.5 and 5 μg ml⁻¹, respectively. Mutagenesis experiments showed that spores exposed to EMS (ethyl methane sulphonate) or UV irradiation (254 nm) could infect lettuce leaves in the presence of up to 100 μg ml⁻¹ CAA. The proportion of infected leaves relative to the number of spores inoculated (infection frequency) was inversely related to the concentration of CAA used, ranging between 0 and 160 per 1 × 10⁶ spores. Resistant mutants (RM) lost their resistance within 1–14 reproduction cycles on CAA-treated plants. Crosses were made between RMxWT isolates and RMxRM isolates with an attempt to obtain stable homozygous resistant off-springs. Such crosses yielded few resistant but unstable progeny isolates. Mutagenic treatments given to hybrid isolates also failed to produce stable resistance. Previous gene sequencing data showed that stable resistance to CAAs is based on a single SNP in the cellulose synthase 3 (CesA3) gene of Plasmopara viticola. Therefore, we sequenced a 582 bp DNA fragment of Ces3A of WT, RM and hybrid isolates of B.lactucae. No mutation in this gene fragment was found. We conclude that mutagenic agents like EMS or UV may induce resistance to CAA in Bremia lactucae but this resistance is not stable and not linked to mutations in CesA3 gene.     

Resistance mechanism to carboxylic acid amide fungicides in the cucurbit downy mildew pathogen Pseudoperonospora cubensis

BACKGROUND: Pseudoperonospora cubensis, the causal oomycete agent of cucurbit downy mildew, is responsible for enormous crop losses in many species of Cucurbitaceae, particularly in cucumber and melon. Disease control is mainly achieved by combinations of host resistance and fungicide applications. However, since 2004, resistance to downy mildew in cucumber has been overcome by the pathogen, thus driving farmers to rely only on fungicide spray applications, including carboxylic acid amide (CAA) fungicides. Recently, CAA‐resistant isolates of P. cubensis were recovered, but the underlying mechanism of resistance was not revealed. The purpose of the present study was to identify the molecular mechanism controlling resistance to CAAs in P. cubensis. RESULTS: The four CesA (cellulose synthase) genes responsible for cellulose biosynthesis in P. cubensis were characterised. Resistant strains showed a mutation in the CesA3 gene, at position 1105, leading to an amino acid exchange from glycine to valine or tryptophan. Cross‐resistance tests with different CAAs indicated that these mutations lead to resistance against all tested CAAs. CONCLUSION: Point mutations in the CesA3 gene of P. cubensis lead to CAA resistance. Accurate monitoring of these mutations among P. cubensis populations may improve/facilitate adequate recommendation/deployment of fungicides in the field. Copyright © 2011 Society of Chemical Industry     

A quick and efficient screen for resistance to iron toxicity in lowland rice

Iron (Fe) toxicity is a major stress to rice in many lowland environments worldwide. Due to excessive uptake of Fe²⁺ by the roots and its acropetal translocation into the leaves, toxic oxygen radicals may form and damage cell structural components, thus impairing physiological processes. The typical visual symptom is the “bronzing” of the rice leaves, leading to substantial yield losses, particularly when toxicity occurs during early vegetative growth stages. The problem is best addressed through genotype improvement, i.e., tolerant cultivars. However, the time of occurrence and the severity of symptoms and yield responses vary widely among soil types, years, seasons, and genotypes. Cultivars resistant in one system may fail when transferred to another. Better targeting of varietal improvement requires selection tools improving our understanding of the resistance mechanisms and strategies of rice in the presence of excess iron. A phytotron study was conducted to develop a screen for seedling resistance to Fe toxicity based on individual plants subjected to varying levels of Fe (0–3000 mg L^–1 Fe supplied as Fe(II)SO₄), stress duration (1–5 d of exposure), vapor‐pressure deficit (VPD; 1.1 and 1.8 kPa), and seedling age (14 and 28 d). Genotypes were evaluated based on leaf‐bronzing score and tissue Fe concentrations. A clear segregation of the genotypic tolerance spectrum was obtained when scoring 28 d old seedlings after 3 d of exposure to 2000 mg L^–1 Fe in a high‐VPD environment. In most cases, leaf‐bronzing scores were highly correlated with tissue Fe concentration (visual differentiation in includer and excluder types). The combination of these two parameters also identified genotypes tolerating high levels of Fe in the tissue while showing only few leaf symptoms (tolerant includers). The screen allows selecting genotypes with low leaf‐bronzing score as resistant to Fe toxicity, and additional analyses of the tissue Fe concentration of those can identify the general adaptation strategy to be utilized in breeding programs.     

Potential of Borago officinalis, Sinapis alba L. and Phacelia boratus for Phytoextraction of Cd and Pb from Soil

Heavy metal phytoextraction is a soil remediation technique, which makes use of plants in removing contamination from soil. The plants must thus be tolerant to heavy metals, adaptable to soil and climate characteristics, and able to take up large amounts of heavy metals. Most of the high biomass productive plants such as, maize, oat and sunflower are plants, which do not grow in cold climates or need intensive care. In this study three “weed” plants, Borago officinalis; Sinapis alba L. and Phacelia boratus were investigated for their ability to tolerate and accumulate high amounts of Cd and Pb. Pot experiments were performed with soil containing Cd and Pb at concentrations of up to 180 mg kg^?1 and 2,400 mg kg^?1 respectively. All three plants showed high levels of tolerance. Borago officinalis; and Sinapis alba L. accumulated 109 mg kg^?1 and 123 mg kg^?1 Cd, respectively at the highest Cd spiked soil concentration. Phacelia boratus reached a Cd concentration of 42 mg kg^?1 at a Cd soil concentration of 100 mg kg^?1. In the case of Pb, B. officinalis and S. alba L. displayed Pb concentrations of 25 mg kg^?1 and 29 mg kg^?1, respectively at the highest Pb spiked soil concentration. Although the Pb uptake in P. boratus reached up to 57 mg kg^?1 at a Pb spiked soil concentration of 1,200 mg kg^?1, it is not suitable for phytoextraction because of its too low biomass.