Moon A.F.,National Health Research Institute |
Mueller G.A.,National Health Research Institute |
Zhong X.,National Health Research Institute |
Zhong X.,Syngenta Biotechnology |
Pedersen L.C.,National Health Research Institute
Protein Science | Year: 2010
Protein crystallographers are often confronted with recalcitrant proteins not readily crystallizable, or which crystallize in problematic forms. A variety of techniques have been used to surmount such obstacles: crystallization using carrier proteins or antibody complexes, chemical modification, surface entropy reduction, proteolytic digestion, and additive screening. Here we present a synergistic approach for successful crystallization of proteins that do not form diffraction quality crystals using conventional methods. This approach combines favorable aspects of carrier-driven crystallization with surface entropy reduction. We have generated a series of maltose binding protein (MBP) fusion constructs containing different surface mutations designed to reduce surface entropy and encourage crystal lattice formation. The MBP advantageously increases protein expression and solubility, and provides a streamlined purification protocol. Using this technique, we have successfully solved the structures of three unrelated proteins that were previously unattainable. This crystallization technique represents a valuable rescue strategy for protein structure solution when conventional methods fail. Published by Wiley-Blackwell. © 2010 The Protein Society.
Que Q.,Syngenta Biotechnology
GM crops | Year: 2010
In recent years, there has been a rapid increase in the planting of transgenic crops with stacked traits. Most of these products have been formed by conventional breeding, i.e. the crossing of transgenic plant (event) containing individual transgenes with other event(s) containing single or double transgenic traits. Many biotech companies are developing stacked trait products with increasing numbers of insect and herbicide tolerance genes for controlling a broad range of insect pests and weeds. There has also been an increase in development of technologies for molecular stacking of multiple traits in a single transgene locus. In this review we look at the status of stacked trait products, crop trait stacking technologies and the technical challenges we are facing. We also review recent progress in developing technology for assembling large transgene arrays in vitro (molecular stacks), their delivery to crop plants and issues they pose for transgene expression.
Kelliher T.,Stanford University |
Kelliher T.,Syngenta Biotechnology |
Walbot V.,Stanford University
Plant Journal | Year: 2014
In flowering plants, anthers are the site of de novo germinal cell specification, male meiosis, and pollen development. Atypically, anthers lack a meristem. Instead, both germinal and somatic cell types differentiate from floral stem cells packed into anther lobes. To better understand anther cell fate specification and to provide a resource for the reproductive biology community, we isolated cohorts of germinal and somatic initials from maize anthers within 36 h of fate acquisition, identifying 815 specific and 1714 significantly enriched germinal transcripts, plus 2439 specific and 2112 significantly enriched somatic transcripts. To clarify transcripts involved in cell differentiation, we contrasted these profiles to anther primordia prior to fate specification and to msca1 anthers arrested in the first step of fate specification and hence lacking normal cell types. The refined cell-specific profiles demonstrated that both germinal and somatic cell populations differentiate quickly and express unique transcription factor sets; a subset of transcript localizations was validated by in situ hybridization. Surprisingly, germinal initials starting 5 days of mitotic divisions were enriched significantly in >100 transcripts classified in meiotic processes that included recombination and synapsis, along with gene sets involved in RNA metabolism, redox homeostasis, and cytoplasmic ATP generation. Enrichment of meiotic-specific genes in germinal initials challenges current dogma that the mitotic to meiotic transition occurs later in development during pre-meiotic S phase. Expression of cytoplasmic energy generation genes suggests that male germinal cells accommodate hypoxia by diverting carbon away from mitochondrial respiration into alternative pathways that avoid producing reactive oxygen species (ROS). © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.
Guo Z.,Syngenta Biotechnology
TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik | Year: 2014
Impacts of population structure on the evaluation of genomic heritability and prediction were investigated and quantified using high-density markers in diverse panels in rice and maize. Population structure is an important factor affecting estimation of genomic heritability and assessment of genomic prediction in stratified populations. In this study, our first objective was to assess effects of population structure on estimations of genomic heritability using the diversity panels in rice and maize. Results indicate population structure explained 33 and 7.5% of genomic heritability for rice and maize, respectively, depending on traits, with the remaining heritability explained by within-subpopulation variation. Estimates of within-subpopulation heritability were higher than that derived from quantitative trait loci identified in genome-wide association studies, suggesting 65% improvement in genetic gains. The second objective was to evaluate effects of population structure on genomic prediction using cross-validation experiments. When population structure exists in both training and validation sets, correcting for population structure led to a significant decrease in accuracy with genomic prediction. In contrast, when prediction was limited to a specific subpopulation, population structure showed little effect on accuracy and within-subpopulation genetic variance dominated predictions. Finally, effects of genomic heritability on genomic prediction were investigated. Accuracies with genomic prediction increased with genomic heritability in both training and validation sets, with the former showing a slightly greater impact. In summary, our results suggest that the population structure contribution to genomic prediction varies based on prediction strategies, and is also affected by the genetic architectures of traits and populations. In practical breeding, these conclusions may be helpful to better understand and utilize the different genetic resources in genomic prediction.
Raybould A.,Hill International |
Kilby P.,Hill International |
Graser G.,Syngenta Biotechnology
Transgenic Research | Year: 2013
Most commercial transgenic crops are genetically engineered to produce new proteins. Studies to assess the risks to human and animal health, and to the environment, from the use of these crops require grams of the transgenic proteins. It is often extremely difficult to produce sufficient purified transgenic protein from the crop. Nevertheless, ample protein of acceptable purity may be produced by over-expressing the protein in microbes such as Escherichia coli. When using microbial proteins in a study for risk assessment, it is essential that their suitability as surrogates for the plant-produced transgenic proteins is established; that is, the proteins are equivalent for the purposes of the study. Equivalence does not imply that the plant and microbial proteins are identical, but that the microbial protein is sufficiently similar biochemically and functionally to the plant protein such that studies using the microbial protein provide reliable information for risk assessment of the transgenic crop. Equivalence is a judgement based on a weight of evidence from comparisons of relevant properties of the microbial and plant proteins, including activity, molecular weight, amino acid sequence, glycosylation and immuno-reactivity. We describe a typical set of methods used to compare proteins in regulatory risk assessments for transgenic crops, and discuss how risk assessors may use comparisons of proteins to judge equivalence. © 2012 The Author(s).