Genetic engineering最新文献

The OPT family was first described six years ago, and much progress has been made in understanding the role these transporters play in their respective organisms. Plants are the only organisms in which both YS- and PT-type transporters have been characterized, and all of these OPTs appear to be plasma membrane-bound proteins, suggesting that they import substrates from the apoplasm or the environment. YS1 is the only OPT known to translocate substrates from the rhizosphere, whereas all the other OPTs seem to function in long-distance transport of peptides or metals. The sum of all the studies covered in this review suggest the model for OPT function in plants depicted in Figure 4. Peptides, metal-NA, and metal-MAs complexes (Strategy II plants only) are loaded into the xylem stream in the root for long-distance transport. OPTs unload the xylem by importing substrates into sink tissues such as leaves and by transloading the phloem. Peptides and metal-NA complexes exit the leaf symplasmically or by importation into the phloem from the apoplasm by OPTs. The filial tissues (endosperm and embryo) are apoplasmically separated from the maternal tissues, and OPTs may also function in loading the developing seed. Similarly, seedlings are symplasmically disconnected from the endosperm and OPTs may help move nutrients to the growing plant. Much progress has been made in the last two years toward understanding OPTs in plants, although several fundamental questions remain unanswered. Namely, what is the level of redundancy? Is there any substrate overlap between YS and PT OPTs? How crucial are their respective roles? Are there additional functions beyond peptide and metal transport? Given the recent pace of discovery, we may not have to wait long to find out the answers.

High-throughput approaches for gene cloning and expression require the development of new, nonstandard tools for use by molecular biologists and biochemists. We have developed and implemented a series of methods that enable the production of expression constructs in 96-well plate format. A screening process is described that facilitates the identification of bacterial clones expressing soluble protein. Application of the solubility screen then provides a plate map that identifies the location of wells containing clones producing soluble proteins. A series of semi-automated methods can then be applied for validation of solubility and production of freezer stocks for the protein production group. This process provides an 80% success rate for the identification of clones producing soluble protein and results in a significant decrease in the level of effort required for the labor-intensive components of validation and preparation of freezer stocks. This process is customized for large-scale structural genomics programs that rely on the production of large amounts of soluble proteins for crystallization trials.

Long-term potentiation (LTP) and long-term depression (LTD) are two experimental models of synaptic plasticity that have been studied extensively in the last 25 years, as they may represent basic mechanisms to store certain types of information in neuronal networks. In several brain regions, these two forms of synaptic plasticity require dendritic depolarization, and the amplitude and duration of the depolarization-induced calcium signal are crucial parameters for the generation of either LTP or LTD. The rise in calcium concentration mediated by activation of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors has been proposed to stimulate various calcium-dependent processes that could convert the induction signal into long-lasting changes in synaptic structure and function. According to several lines of experimental evidence, alterations in synaptic function observed with LTP and LTD are thought to be the result of modifications of postsynaptic currents mediated by the a-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) subtype of glutamate receptors. The question of which type(s) of receptor changes constitutes the basis for the expression of synaptic plasticity is still very much open. Here, we review data relevant to the issue of selective modulation of AMPA receptor properties occurring after learning and memory, environmental enrichment, and synaptic plasticity. We also discuss potential cellular mechanisms whereby calcium-dependent enzymes might regulate AMPA receptor properties during LTP and LTD, focusing on protein kinases, proteases and lipases.