Characterization of a signal peptide sequence in the cell-free translation product of sheep elastin mRNA

In vitro explant cultures of near-term sheep nuchal ligament secrete tropoelastin of approximate M_r 70,000–72,000 while the elastin cell-free product of sheep nuchal ligament RNA is 2000 to 3000 M_r larger. Automated Edman degradation of immunoprecipitates of radiolabeled cell-free elastin precursor demonstrated the presence of a 26-residue signal sequence which was absent from sheep tropoelastin secreted from explant cultures. In addition, a 20-residue overlap was established between the cell-free product and the secreted protein. This overlap region, representing the N-terminal sequence of ovine tropoelastin, demonstrated complete homology with the N-terminal sequence of porcine tropoelastin and near complete homology with chick tropoelastin. These findings suggest that cotranslational removal of this hydrophobic peptide extension is likely a correlate of vectorial transport of elastin into the secretory apparatus.     

Dynamics of endoglucanase catalytic domains: implications towards thermostability

Thermostable endoglucanases play a crucial role in the production of biofuels to breakdown plant cellulose. Analyzing their structure-dynamics relationship can inform about the origins of their thermostability. Although tertiary structures of many endoglucanase proteins are available, the relationship between thermostability, structure, and dynamics is not explored fully. We have generated elastic network models for thermostable and mesostable endoglucanases with the (αβ)? fold in substrate bound and unbound states. The comparative analyses shed light on the relation between protein dynamics, thermostability, and substrate binding. We observed specific differences in the dynamic behavior of catalytic residues in slow modes: while both the nucleophile and the acid/base donor residues show positively correlated motions in the thermophile, their dynamics is uncoupled in the mesophile. Our proof-of-concept comparison study suggests that global dynamics can be harnessed to further our understanding of thermostability.     

Characterization of Cry34/Cry35 binary insecticidal proteins from diverse Bacillus thuringiensis strain collections

Bacillus thuringiensis crystal proteins of the Cry34 and Cry35 classes function as binary toxins showing activity on the western corn rootworm, Diabrotica virgifera virgifera LeConte. We surveyed 6,499 B. thuringiensis isolates by hybridization for sequences related to cry35A genes, identifying 78 strains. Proteins of the appropriate molecular mass (ca. 44 kDa) for Cry35 were observed in 42 of the strains. Full-length, or nearly full-length, sequences of 34 cry34 genes and 16 cry35 genes were also obtained from cloning, PCR analysis, and DNA sequencing. These included representatives of all known Cry34A, Cry34B, Cry35A, and Cry35B classes, as well as a novel Cry34A/Cry35A-like pair. Bioassay analysis indicated that cry35-hybridizing strains not producing a ca. 14-kDa protein, indicative of Cry34, were not active on corn rootworms, and that the previously identified Cry34A/Cry35A pairs were more active than the Cry34B/Cry35B pairs. The cry35-hybridizing B. thuringiensis strains were found in locales and materials typical for other B. thuringiensis strains. Comparison of the sequences with the geographic origins of the strains showed that identical, or nearly identical, sequences were found in strains from both Australasia and the Americas. Sequence similarity searches revealed that Cry34 proteins are similar to predicted proteins in Photorhabdus luminescens and Dictyostelium discoidium, and that Cry35Ab1 contains a segment similar to beta-trefoil domains that may be a binding motif. The binary Cry34/Cry35 B. thuringiensis crystal proteins thus appear closely related to each other, are environmentally ubiquitous, and share sequence similarities consistent with activity through membrane disruption in target organisms.     

Representativity of a mesoscale network for weather-related factors governing pollen dispersal

The cultivation of transgenic crops, such as maize, requires successful gene isolation in field environments. Five spatial statistical techniques are used to evaluate the use of a regional mesoscale observation network (Iowa Environmental Mesonet) as a means to drive field-scale pollen dispersion modeling. The Nearest Neighbor Index, Fractal Dimension, Morisita Index, Thiessen Polygons, and Coefficient of Representativity are computed showing the positive and negative impacts of sequential addition of observation networks into a mesonet framework (a collection of pre-existing networks). While it is shown that the arbitrary combination of disparate observing networks increases spatial resolution, this improvement is often at the expense of increased clustering due to co-location of observation sites near urban areas. Network composition in terms of density and degree of clustering was evaluated with a grid analysis using the Barnes scheme as a means to mitigate clustering and improve prediction accuracies when mesonet data are applied to modeling. This paper shows the importance of understanding and accounting for the spatial characteristics of an observational network before applying it to a modeling effort such as field scale pollen dispersion.     

Assessment of risk of insect-resistant transgenic crops to nontarget arthropods

An international initiative is developing a scientifically rigorous approach to evaluate the potential risks to nontarget arthropods (NTAs) posed by insect-resistant, genetically modified (IRGM) crops. It adapts the tiered approach to risk assessment that is used internationally within regulatory toxicology and environmental sciences. The approach focuses on the formulation and testing of clearly stated risk hypotheses, making maximum use of available data and using formal decision guidelines to progress between testing stages (or tiers). It is intended to provide guidance to regulatory agencies that are currently developing their own NTA risk assessment guidelines for IRGM crops and to help harmonize regulatory requirements between different countries and different regions of the world.     

Primary structure of a chick tropoelastin peptide: Evidence for a collagen-like amino acid sequence

A 73-residue tryptic fragment from chick aortic tropoelastin has been sequenced by solid state methods. This is a new structure not previously described in any tropoelastins and may be unique for avians. It contains a G V P tripeptide repeat, giving it a primary structure like that of collagen. This may explain some of the collagen-like properties observed in elastin, particularly collagenase susceptibility and the presence of hydroxyproline.     

Genetically modified crops for the bioeconomy: meeting public and regulatory expectations

As the United States moves toward a plant-based bioeconomy, a large research and development effort is focused on creating new feedstocks to meet biomass demand for biofuels, bioenergy, and specialized bioproducts, such as industrial compounds and biomaterial precursors. Most bioeconomy projections assume the widespread deployment of novel feedstocks developed through the use of modern molecular breeding techniques, but rarely consider the challenges involved with the use of genetically modified crops, which can include hurdles due to regulatory approvals, market adoption, and public acceptance. In this paper we consider the implications of various transgenic crops and traits under development for the bioeconomy that highlight these challenges. We believe that an awareness of the issues in crop and trait selection will allow developers to design crops with maximum stakeholder appeal and with the greatest potential for widespread adoption, while avoiding applications unlikely to meet regulatory approval or gain market and public acceptance. The views presented here are those of the authors and do not necessarily represent the views of the US government.     

Advancing environmental risk assessment for transgenic biofeedstock crops

Transgenic modification of plants is a key enabling technology for developing sustainable biofeedstocks for biofuels production. Regulatory decisions and the wider acceptance and development of transgenic biofeedstock crops are considered from the context of science-based risk assessment. The risk assessment paradigm for transgenic biofeedstock crops is fundamentally no different from that of current generation transgenic crops, except that the focus of the assessment must consider the unique attributes of a given biofeedstock crop and its environmental release. For currently envisioned biofeedstock crops, particular emphasis in risk assessment will be given to characterization of altered metabolic profiles and their implications relative to non-target environmental effects and food safety; weediness and invasiveness when plants are modified for abiotic stress tolerance or are domesticated; and aggregate risk when plants are platforms for multi-product production. Robust risk assessments for transgenic biofeedstock crops are case-specific, initiated through problem formulation, and use tiered approaches for risk characterization.     

Current risk assessment approaches for environmental and food and feed safety assessment

Foundational activities at the international level underlie current risk and safety assessment approaches for genetically engineered/modified organisms (GEOs/GMOs). Early risk assessment considerations beginning with the OECD ‘Blue Book’ established risk/safety assessment as the characterization of the organism and its environmental release; establishment and persistence in the environment; and human and ecological effects, analyzed in principle through existing methods. Important in this context was recognition that GEOs/GMOs as a class did not represent new risks relative to products of traditional plant breeding and that any incremental risk would need to be established on a stepwise case-by-case comparative basis with existing crops and derived-foods as the baseline. Accordingly, concepts of familiarity and substantial equivalence were advanced by OECD and WHO as ways to establish a risk analysis baseline for determining whether and to what extent risk/safety assessment was needed. Regulatory implementations of this paradigm have skewed to increasingly complex portfolios of studies rather than adhering to analysis which is formulated to fit the risk/safety questions relevant to a given case. Plants produced through genome editing technology will benefit from risk analysis that implements sound problem formulation to guide the need for and nature of risk/safety assessments.