Mutational analysis of highly conserved aspartate residues essential to the catalytic core of the piggyBac transposase

<p>Abstract</p> <p>Background</p> <p>The <it>piggyBac </it>mobile element is quickly gaining popularity as a tool for the transgenesis of many eukaryotic organisms. By studying the transposase which catalyzes the movement of <it>piggyBac</it>, we may be able to modify this vector system to make it a more effective transgenesis tool. In a previous publication, Sarkar A, Sim C, Hong YS, Hogan JR, Fraser MJ, Robertson HM, and Collins FH have proposed the presence of the widespread 'DDE/DDD' motif for <it>piggyBac </it>at amino acid positions D268, D346, and D447.</p> <p>Results</p> <p>This study utilizes directed mutagenesis and plasmid-based mobility assays to assess the importance of these residues as the catalytic core of the <it>piggyBac </it>transposase. We have functionally analyzed individual point-mutations with respect to charge and physical size in all three proposed residues of the 'DDD' motif as well as another nearby, highly conserved aspartate at D450. All of our mutations had a significant effect on excision frequency in S2 cell cultures. We have also aligned the <it>piggyBac </it>transposase to other close family members, both functional and non-functional, in an attempt to identify the most highly conserved regions and position a number of interesting features.</p> <p>Conclusion</p> <p>We found all the designated DDD aspartates reside in clusters of amino acids that conserved among <it>piggyBac </it>family transposase members. Our results indicate that all four aspartates are necessary, to one degree or another, for excision to occur in a cellular environment, but D450 seems to have a tolerance for a glutamate substitution. All mutants tested significantly decreased excision frequency in cell cultures when compared with the wild-type transposase.</p

Plant community attributes affect dry grassland orchid establishment

Several factors have been taken into account to explain the distribution of orchid species. We explored the extent to which plant community attributes affect the abundance and reproductive fitness of three orchid species (Anacamptis morio, Himantoglossum adriaticum and Ophrys sphegodes), native to dry grasslands. Structural attributes of plant community (e.g. cover and height) were assessed in ninety 4 m(2) plots scattered on three hill massifs of the Veneto Region (NE Italy). For the three target orchid species, the height of the flowering stalk, the relative ramet height and the number of flowers and fruits were recorded in 203 tagged ramets. Generalized Linear Model revealed that plant community attributes such as cover and height of the herb layer exert a negative effect on the abundance of orchid populations. Furthermore, regression models indicated that O. sphegodes and H. adriaticum reproductive fitness, determined as fruit/flower ratio, was positively affected by relative ramet height. Our results revealed that local herbaceous vegetation structure influences the cover and fruit set of target orchid species. However, there can be substantial variation in the response of different species and variation in the structural attributes of surrounding vegetation may be associated with differences in the strength of selection. In order to achieve effective results in orchid species conservation, protocols for the in situ conservation must detail the range of vegetation covers and heights at which orchid species are favoured and can produce the most effective inflorescences

A FUNCTIONAL ANALOGY BETWEEN CROWN ETHERS AND METALLACROWNS

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DEVELOPMENT OF METALLOCROWN ETHERS

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