T here are many types of microscopy, each providing a unique set of benefits. Light microscopy of cells that express fluorescently labelled molecules, for example, lets scientists observe the movements of specific molecules or protein complexes in live cells in real time, or in fixed samples. Scanning electron microscopy (EM) reveals tiny details of the cell surface, and transmission EM shows the detailed cytoarchitecture of sections through fixed tissue. Other aspects of the structure of cells and tissues can be explored using techniques such as ion microscopy, total internal reflection microscopy, atomic force microscopy and super-resolution microscopy. Each type provides different information, but using two sorts of microscopy simultaneously provides even more -and molecular tools have been developed to link them together. Researchers testing the waters of this 'correlative microscopy' are beginning to discover its challenges and rewards.Correlated light-electron microscopy, for example, provides both the specificity and real-time observation of light microscopy with fluorescent labelling and the better structural resolution of EM. But such correlative microscopy has historically been tricky to use. Scientists who tried it might have needed to switch to EM halfway through an experiment, which meant moving the sample from one This image of a butterfly wing was constructed using two types of microscopy: a confocal image of the reflective eyespots shows scales (green) and wing (red); scanning electron microscopy reveals the different structure of non-reflective scales (upper left).

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To better understand the potential for antagonistic interactions between members of the same bacterial species, we have surveyed bacteriocin killing activity across a diverse suite of strains of the phytopathogen Pseudomonas syringae. Our data demonstrate that killing activity from phage-derived bacteriocins of P. syringae (R-type syringacins) is widespread. Despite a high overall diversity of bacteriocin activity, strains can broadly be classified into five main killing types and two main sensitivity types. Furthermore, we show that killing activity switches frequently between strains and that switches correlate with localized recombination of two genes that together encode the proteins that specify bacteriocin targeting. Lastly, we demonstrate that phage-derived bacteriocin killing activity can be swapped between strains simply through expression of these two genes in trans. Overall, our study characterizes extensive diversity of killing activity for phage-derived bacteriocins of P. syringae across strains and highlights the power of localized recombination to alter phenotypes that mediate strain interactions during evolution of natural populations and communities.

or the pharmaceutical industry, the Human Genome Project has proved to be both a blessing and a curse. Where potential drug targets were once hard to come by, the industry is now awash with them. This has left researchers with the unenviable challenge of sifting through the data in search of the elusive proteins that are instrumental in human disease. Akin to seeking a needle in a haystack, this herculean task has boosted the importance of rapid screening technologies. Of the roughly 35,000 genes in the human genome, only a few have known functions. So the task of identifying and verifying a positive lead is key to effective drug development. Drugs fail in the clinic for two basic reasons: they either don't work or they prove to be unsafe. "Both of these are often the direct result of sloppy early target validation," says David Szymkowski, director of biotherapeutics at the biopharmaceutical company Xencor in Monrovia, California. Validation is a crucial step in the drugdiscovery process. Most drugs are inhibitors that block the action of a particular target drug target validation technology feature Hitting the target For any protein, it is not guaranteed that all of its isoforms will have the same function, so it is important to work out which forms are valid drug targets. Many proteins are expressed as different isoforms as a result of alternative splicing of the precursor messenger RNAs (mRNAs) and post-translational modifications. Techniques such as gene knockouts effectively remove all isoforms, but manipulation of mRNA with antisense technology or RNA interference (see 'The silent treatment', page 343) cannot distinguish between isoforms that differ in their post-translational modifications. To overcome this problem, biotechnology company Sangamo BioSciences in Richmond, California, has developed a system that allows the expression of endogenous genes to be altered in cells or whole-animal models. It uses zinc-finger transcription factors in which the DNA-recognition domain is coupled to a functional domain that allows the expression of the target gene to be up-or downregulated. The advantage of this method over gene knockouts or transgenes is that the expression levels of all the isoforms of an endogenous target gene can be specifically manipulated. This technique has been used by a group including researchers at Sangamo and Frank Giordano at Yale University to investigate the growth of blood vessels by activating the endogenous expression of vascular endothelial growth factor (VEGF). The team found that all isoforms of VEGF are needed to stimulate the growth of blood vessels that do not leak (E. J. Rebar et al. Nature Med. 8, 1427-1432; 2002). Casey Case of Sangamo is excited by what he sees as the growing awareness of the biological importance of alternative mRNA splicing. "Conventional overexpression methods for target validation miss the biological consequences of this important phenomenon," he says. Another approach to the problem has been developed by Xerion Pharmaceuticals in Martinsried, Germany. I...

To better understand the potential for detrimental interactions between strains of the same bacterial species, we have surveyed bacteriocin killing activity across a diverse suite of strains of the phytopathogen Pseudomonas syringae. Our data demonstrate that killing activity from phage derived bacteriocins of P. syringae (R-type syringacins) is widespread. Despite a high overall diversity of bacteriocin activity, strains can broadly be classified into five main killing types and two main sensitivity types. Furthermore, we show that killing activity switches frequently between strains, and that switches correlate with localized recombination of two genes that together encode the proteins that specify bacteriocin targeting. Lastly, we demonstrate that phage derived bacteriocin killing activity can be swapped between strains simply through expression of these two genes in trans. Overall, our study characterizes extensive diversity of killing activity for phage derived bacteriocins of P. syringae across strains and highlights the power of localized recombination to alter phenotypes that mediate strain interactions during evolution of natural populations and communities.