Use of green fluorescent protein as A non-destructive marker for peanut genetic transformation

Summary The ability to non-destructively visualize transient and stable gene expression has made green fluorescent protein (GFP) a most efficient reporter gene for routine plant transformation studies. We have assessed two fluorescent protein mutants, enhanced GFP (EGFP) and enhanced yellow fluorescent protein (EYFP), under the control of the CaMV35S promoter, for their transient expression efficiencies after particle bombardment of embryogenic cultures of the peanut cultivar, Georgia Green. A third construct (p524EGFP.1) that expressed EGFP from a double 35S promoter with an AMV enhancer sequence also was compared. The brightest and most dense fluorescent signals observed during transient expression were from p524EGFP. 1 and EYFP. Optimized bombardment conditions consisted of 0.6 μm diameter gold particles, 12410 kPa bombardment pressure, 95 kPa vacuum pressure, and pretreatment with 0.4 M mannitol. Bombardments with p524EGFP.1 produced tissue sectors expressing GFP that could be visually selected under the fluorescence microscope over multiple subcultures. Embryogenic lines selected for GFP expression initially may have been chimeric since quantitative analysis of expression sometimes showed an increase when GFP-expressing lines, that also contained a hygromycin-resistance gene, subsequently were cultured on hygromycin. Transformed peanut plants expressing GFP were obtained from lines selected either visually or on hygromycin. Integration of the gfp gene in the genomic DNA of regenerated plants was confirmed by Southern blot hybridization and transmission to progeny.     

Oviposition preference for novel versus normal food resources in laboratory populations ofDrosophila melanogaster

Female fecundity, oviposition preference and specificity on one normal and two novel food media were assayed on four laboratory populations ofDrosophila melanogaster, revealing considerable among- and within-population variation in oviposition preference. Overall, there was a significant tendency of females to prefer novel media to their normal banana food as an oviposition substrate. Specificity in the populations was fairly high, implying that a large proportion of females tended to lay the majority of their eggs on the preferred medium. The results showed that oviposition preference for a given food medium could be affected by the alternative provided, and that, consequently, oviposition preference for a given food medium versus another cannot be predicted based upon a knowledge of what the preference for each of the two media was versus a common third medium. Specificity, on the other hand, was not significantly affected by the type of alternative food media provided in a given trial. Moreover, comparison of results from fecundity and oviposition preference assays also showed that the egg laying behaviour ofDrosophila females in response to different food media may be different in choice versus no-choice situations. Thus, a substrate on which fecundity is higher than on another, when assayed in a no-choice situation, may not be preferred over the other substrate when a choice between the two is provided to the ovipositing females. The latter two results point to possible complexity in the responses of females to various oviposition substrates based upon the overall setting of the assay, including the alternative substrates present for egg laying.     

Antidiabetic activity of aqueous extract and non polysaccharide fraction of Cynodon dactylon Pers

Petroleum ether (60 degrees-80 degrees C), chloroform, acetone, ethanol, aqueous and crude hot water extracts of the whole plant of C. dactylon and the two fractions of aqueous extract were tested for antihyperglycaemic activity in glucose overloaded hyperglycemic rats and in alloxan induced diabetic model at two-dose levels, 200 and 400 mg/kg (po) respectively. The aqueous extract of C. dactylon and the non polysaccharide fraction of aqueous extract were found to exhibit significant antihyperglycaemic activity and only the non polysaccharide fraction was found to produce hypoglycemia in fasted normal rats. Treatment of diabetic rats with aqueous extract and non polysaccharide fraction of the plant decreased the elevated biochemical parameters, glucose, urea, creatinine, serum cholesterol, serum triglyceride, high density lipoprotein, low density lipoprotein, haemoglobin and glycosylated haemoglobin significantly. Comparatively, the non polysaccharide fraction of aqueous extract was found to be more effective than the aqueous extract.     

Bioprospecting in plants for engineered proteins

For more than two decades, bioengineered plants have produced protein therapeutics for human and animal use. Almost all proteins produced by other existing systems, including antibodies, vaccines and plasma proteins, have now been manufactured in plants. Considering the limitations of microbial and mammalian reactor-based protein-production technologies and the impending bottleneck in manufacturing capacity, plants are now emerging as an attractive alternative system with which to supply the growing need for protein-based therapeutics. However, full realization of the promise of plant-derived engineered proteins requires that we confront the dual challenges of bioequivalence and product consistency, challenges that are largely related to post-translational protein modifications (PTMs) that are crucial to the structure and function of most eukaryotic proteins. Among the protein PTMs, the foremost challenge for bioactivity and acceptance by the pharmaceutical and biotechnology industries and regulatory agencies is glycosylation. Advances made in recent years that 'humanize' plant glycosylation pathways combined with the discovery of terminal sialic acids (SAs) in plants now make feasible the bioengineering in plants of glycoproteins that have mammalian-like glycosylation.     

Direct Expression and Validation of Phage-selected Peptide Variants in Mammalian Cells

Phage display is a key technology for the identification and maturation of high affinity peptides, antibodies, and other proteins. However, limitations of bacterial expression restrict the range and sensitivity of assays that can be used to evaluate phage-selected variants. To address this problem, selected genes are typically transferred to mammalian expression vectors, a major rate-limiting step in the iterative improvement of peptides and proteins. Here we describe a system that combines phage display and efficient mammalian expression in a single vector, pDQ1. This system permits immediate expression of phage-selected genes as IgG1-Fc fusions in mammalian cells, facilitating the rapid, sensitive characterization of a large number of library outputs for their biochemical and functional properties. We demonstrate the utility of this system by improving the ability of a CD4-mimetic peptide to bind the HIV-1 envelope glycoprotein and neutralize HIV-1 entry. We further improved the potency of the resulting peptide, CD4mim6, by limiting its ability to induce the CD4-bound conformation of the envelope glycoprotein. Thus, CD4mim6 and its variants can be used to investigate the properties of the HIV-1 envelope glycoprotein, and pDQ1 can accelerate the discovery of new peptides and proteins through phage display.     

Evolution of faster development does not lead to greater fluctuating asymmetry of sternopleural bristle number inDrosophila

Both strong directional selection and faster development are thought to destabilize development, giving rise to greater fluctuating asymmetry (FA), although there is no strong empirical evidence supporting this assertion. We compared FA in sternopleural bristle number in four populations ofDrosophila melanogaster successfully selected for faster development from egg to adult, and in four control populations. The fraction of perfectly symmetric individuals was higher in the selected populations, whereas the FA levels did not differ significantly between selected and control populations, clearly indicating that directional selection for faster development has not led to increased FA in sternopleural bristle number in these populations. This may be because: (i) development time and FA are uncorrelated, (ii) faster development does result in FA, but selection has favoured developmentally stable individuals that can develop fast and still be symmetrical, or (iii) the increased fraction of symmetric individuals in the selected populations is an artifact of reduced body size. Although we cannot discriminate among these explanations, our results suggest that the relationship between development time, FA and fitness may be far more subtle than often thought.