Recombinant cloning made sexy

Credit: Dennis Kunkel Microscopy, Inc.

Recombinant DNA technologies based on in vitro nucleic acid manipulations using a battery of bacterial enzymes have been the cornerstone of molecular biology for the past several decades. Going back to basics, Li and Elledge now harness the in vivo recombination capabilities of the bacterium Escherichia coli to assist molecular biologists with the task of subcloning genes without having to resort to the sometimes cumbersome and complex manipulations traditionally required. The authors, who have called their approach mating-assisted genetically integrated cloning (MAGIC), have designed a method by which a DNA fragment of interest is transferred from a donor vector in one bacterial strain to a recipient vector in a second bacterial strain through the natural mating and homologous recombination processes inherent to most bacteria. Their donor/recipient vector system provides a platform for the seamless and rapid generation of a variety of constructs, including expression vectors for different organisms. The simplicity of the approach should facilitate high-throughput recombinant DNA manipulation and represents a substantial time and effort savings for researchers in general. (Nat. Gen. 37, 311–319, 2005) GTO

Early Alzheimer detection?

Current tests for Alzheimer disease, based on cognitive and neurological assessments and in vivo brain imaging, are not entirely accurate; a definitive diagnosis can be made only with post-mortem brain samples. A better approach might be to detect protein markers of the disease, such as amyloid-β-diffusable ligands (ADDLs), in cerebrospinal fluid, but the concentrations of these markers are too low to be detected by existing assays. Chad Mirkin and colleagues now report success in detecting ADDLs in cerebrospinal fluid with great accuracy. In their 'sandwich' assay, the biomarker of interest is bound by both a gold nanoparticle and a magnetic microparticle by analyte-specific antibodies attached to both particle types. The magnetic particle allows purification, and the gold particle, which carries hundreds of 'barcode' double-stranded DNA sequences, allows the analyte to be identified. The assay's high sensitivity derives from the amplification achieved by the release of hundreds of DNA strands per single analyte-binding event. As few as tens of copies of a protein can be detected. The team studied ADDL concentrations in 30 individuals, half diagnosed with Alzheimer disease. The diagnosed population showed higher ADDL levels and almost no overlap in concentration ranges with the control population. With further development, this assay may allow earlier and more reliable detection of Alzheimer disease. (Proc. Natl. Acad. Sci. USA 102, 2273–2276, 2005) MZ

Vaccine glycosylation by design

Glycosylation was once thought to be the domain of eukaryotes. However, studies of bacterial antigens, like the O antigen of Gram-negative bacteria, show that glycosylation confers important functions to bacterial proteins. Now Feldman and colleagues find that a single enzyme, PglB, from the food-borne pathogen Campylobacter jejuni, uses the same basic mechanism for glycosylating proteins as the more complex yeast glycosylation apparatus, the oligosaccharyltransferase, a protein complex with no less than nine subunits. Previously, the authors had shown that transferring the pgl locus into Escherichia coli resulted in glycosylation of a membrane protein. In the present work, they demonstrate that the pathway of sugar addition by PglB is identical to the eukaryotic pathway—PglB transfers preformed polysaccharide through a lipid carrier, which in turn transfers the sugars to only certain acceptor sites in a target protein. In addition, they showed that PglB transferred different oligosaccharides, making it more flexible than its eukaryotic counterparts. The authors suggest that the simplicity of the bacterial system makes it more amenable to dissection, and that the relaxed specificity of PglB will make it useful in designing polysaccharide-containing proteins for vaccine production. (Proc. Natl. Acad. Sci. USA 102, 3016–3021, 2005) LD

Good-bye to hearing aids?

Hearing loss has been partly corrected in deafened animals, suggesting a gene-therapy approach for treating some forms of hearing loss in people. Environmental damage to the hair cells of the inner ear—from overstimulation, drugs, infection or simply from aging—destroys hearing irreversibly, as hair cells are not regenerated. Raphael and colleagues asked whether replacement hair cells could be generated by transdifferentiation of other cell types in the auditory epithelium. The Atoh1 gene encodes a transcription factor known to be essential in hair cell development. The authors delivered Atoh1 by adenoviral vector to the cochleae of young-adult guinea pigs whose hair cells had been killed by ototoxic drugs. In a matter of weeks, numerous new hair cells appeared in the organ of Corti, although mixed phenotypes—combining features of hair cells and supporting cells—were also seen. Measurements of auditory brain stem responses showed a significant improvement in the animals' hearing by eight weeks. Beyond their implications for hearing-loss treatment, these results highlight the potential of regenerative strategies based on the expression of developmental genes in adult cells. (Nat. Med. published online 13 February 2005, 10.1038/nm1193) KA

Knocking out malaria

Two reports by Mueller and colleagues suggest that attenuated whole-organism vaccines developed through genetic modification could be used to combat malaria. As part of its complex life cycle, Plasmodium, the malaria parasite, is transmitted to humans and other mammals as sporozoites via mosquito bites. The sporozoites travel to the liver, enter the 'liver stage,' replicate and develop into merozoites, which infect red blood cells. Sporozoites are ideal targets for vaccine development because their eradication would prevent blood infection. Using expression profiling, the researchers had previously found several genes essential for the development of the liver stages of Plasmodium. Two of these genes, UIS3 and UIS4, have now been knocked-out by the authors in P. berghei, a strain that readily infects mice. The resulting parasites are incapable of developing into merozoites, and immunization of mice with UIS-deficient sporozoites results in mice immune to subsequent challenges with wild-type P. berghei sporozoites. These results suggest the possibility of developing similar vaccines against malaria in humans by targeting genes homologous to UIS3 and UIS4 in the human malarial parasite P. falciparum. (Nature 433, 164–167, 2005 & Proc. Natl. Acad. Sci. USA 102, 3022–3027, 2005) TM

Research Highlights written by Kathy Aschheim, Laura DeFrancesco, Teresa Moogan, Gaspar Taroncher-Oldenburg and Mark Zipkin.