Purification and identification of positive regulators binding to a novel element in the c-Jun promoter

A putative response element, GAGCCTC, was observed years ago in footprinting analysis of the c-jun promoter, and here we investigate its function in regulating c-jun expression and identify a protein complex that binds there. Electrophoretic mobility shift assays demonstrate a sequence-specific binding complex with this element in HEK293 cells. Additionally, unlabeled consensus AP-1 element DNA, but not a similar NF-jun element DNA, competes with complex formation. Mutations of this element decrease c-jun promoter reporter activity by nearly 5-fold in HEK293 cells. A new, two-step oligonucleotide trapping technique was developed to purify the element binding proteins. LC-nanospray-ESI-MS/MS identification and Western blotting show that the purified complex contains Ku80 and c-jun, which was further confirmed by antibody supershift, by immunoprecipitation with Southwestern blot or with UV cross-linking analysis in vitro as well as chromatin immunoprecipitation in vivo. c-Jun promoter activity and c-jun expression were decreased by Ku80 siRNA introduction. A mutant Ku80 plasmid with normal amino acid sequence but immune to the siRNA recovers c-jun promoter activity from siRNA inhibition. Similarly, Ku70 wild type transfection can also upregulate c-jun promoter activity. Thus, Ku80-c-jun activates c-jun expression by binding to this GAGCCTC element in the c-jun promoter and Ku70 may also serve a role.     

Interactions of Insecticidal Toxin Gene Products from Xenorhabdus nematophilus PMFI296

Four genes on a genomic fragment from Xenorhabdus nematophilus PMFI296 were shown to be involved in insecticidal activity towards three commercially important insect species. Each gene was expressed individually and in combinations in Escherichia coli, and the insecticidal activity of the lysates was determined. The combined four genes (xptA1, xptA2, xptB1, and xptC1), in E. coli, showed activity towards Pieris brassicae, Pieris rapae, and Heliothis virescens. The genes xptA1, xptB1, and xptC1 were involved in expressing activity towards P. rapae and P. brassicae, while the genes xptA2, xptB1, and xptC1 were needed for activity towards H. virescens. When each of these three genes was expressed individually in E. coli and the cell lysates were used in insect assays or mixed and then used, insecticidal activity was detected at a very low level. If the genes xptB1 and xptC1 were expressed in the same E. coli cell and this cell lysate was mixed with cells expressing xptA1, activity was restored to P. rapae and P. brassicae. Similarly mixing XptB1/C1 lysate with XptA2 lysate restored activity towards H. virescens. Individual gene disruptions in X. nematophilus PMFI296 reduced activity to insects; this activity was restored by complementation with cells expressing either xptA1 or xptA2 for their respective disruptions or E. coli expressing both xptB1 and xptC1 for individual disruptions of either of these genes. The genes xptA2, xptC1, and xptB1 were expressed as an operon in PMFI296 and inactivation of xptA2 or xptC1 resulted in silencing of downstream gene(s), while xptA1 was expressed as a single gene. Therefore, the two three gene product combinations interact with each other to produce good insecticidal activity.     

Effects of carbohydrate availability on lipogenesis in sheep

1. Lipogenesis in sheep liver and adipose tissue was investigated by incorporation studies in vitro with radioactive glucose and acetate and by assays of key enzymes. 2. Carbohydrate availability to sheep was increased by feeding on a diet containing 70% soluble carbohydrate, by infusing glucose into the abomasum or by direct intravenous infusion of glucose. 3. Under these conditions lipogenesis from glucose and acetate was increased from very low values in lìver and adipose tissue, especially in those animals where rumen fermentation was by-passed by glucose infusion. 4. Large increases in the activities of ATP citrate lyase (EC 4.1.3.8) and NADP-malate dehydrogenase (EC 1.1.1.40) occurred in both tissues when lipogenesis was increased. 5. No adaptations were found in the activities of pyruvate carboxylase (EC 6.4.1.1) in adipose tissue, glucokinase (EC 2.7.1.2) in liver or 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) in liver. It is proposed that the absence of these enzymes is not related to glucose availability. 6. The effect of glucose on liver lipogenesis was to increase conversion of acetate into lipid. 7. This effect also occurred in adipose tissue, but in this tissue glucose also became a quantitatively important precursor of triglyceride fatty acid.     

Non‐adaptive phenotypic plasticity: the effects of terrestrial and aquatic herbicides on larval salamander morphology and swim speed

Phenotypic plasticity, although ubiquitous, may not always be advantageous. Non‐adaptive plasticity is likely to occur in response to novel environmental stress. Anthropogenic contaminants, such as herbicides, are novel stressors that are not present in the evolutionary history of most species. We investigated the pattern and consequences of phenotypic plasticity induced by four glyphosate‐based herbicides (two terrestrial and two aquatic) in larvae of the spotted salamander, Ambystoma maculatum, by determining (1) whether the herbicides induced different morphologies; (2) if different morphologies translated to differences in burst swim performance; and (3) how induced individuals performed relative to non‐induced controls. Different herbicide formulations led to the production of significantly different head and tail morphologies, and tail morphology correlated with fastest escape speed. However, escape speed did not vary among treatments. In addition, three out of four herbicide treatments experienced accelerated growth rates, in terms of the lateral size of tails, although the tail shapes were either similar to preliminary controls or intermediate between preliminary and final controls. These observations suggest that herbicide‐induced morphology is a case of non‐adaptive phenotypic plasticity, and that there is potentially a trade‐off between growth and development for larvae exposed to different formulations. Understanding the functional significance of induced phenotypes is important for determining their importance in shaping an organism's ecological interactions and evolutionary trajectories. Furthermore, under different conditions, the morphological changes that we observed in response to exposure to herbicides might affect salamander fitness and influence population dynamics.