It would be fair to say that S. cerevisiae is the model eukaryotic model organism. Safe, fast growing, amenable to genetic manipulation, and with a host of databases and community resources, yeast offer a wealth of opportunities for deciphering gene function and metabolic pathways. Given the significant gene homology between yeast and other frequently studied organisms, even researchers who do not work with yeast directly will likely make use of information gathered in S. cerevisiae. Therefore, an overview of the tools and technologies employed in yeast based functional genomics and proteomics, as provided by Surer et al. on p. 625, is indispensable reading. The review balances discussions of established yeast resources such as the yeast deletion strain collection with up and coming techniques such as eMAP. Other topics covered include drug based screens, large-scale protein localization studies, interactomics, and protein microarrays. In addition, the authors devote considerable attention to the many flavors and applications of the yeast two hybrid system. It's a gargantuan task that Suter et al. handle with aplomb, producing a review that is both comprehensive and eminently readable, thanks to a strong emphasis on the practical strengths and shortcomings of each methodology.
Bait and Snip
Affinity columns have been around for many years, as have immobilized protease reactor columns. The latter are used to cleave precursor molecules into an active form by passing over a column containing immobilized enzymes. Now, Shimizu and Hasumi (p. 590) have combined these two concepts to create a facile means to proteolytically remove and isolate an active cleavage product from its precursor. The advantage of this affinity capture reactor system is that it allows initial concentration on the column of the substrate from a crude mixture, followed by cleavage under conditions that are more favorable for maintaining substrate activity and preventing denaturation. Final purification of the cleaved product is also simplified. To demonstrate the application of the system, the authors covalently immobilized lysine, which binds to plasminogen, plus the metalloproteinase bacillolysin MA (BL-MA) on a sepharose column, over which was passed crude human plasma. Plasminogen in the plasma bound to the immobilized lysine, resulting in retention of these molecules. BL-MA preferentially cleaved the plasminogen at a specific site to release the active angiostatin product, which was then eluted. High purity product with a good recovery rate could be obtained, and the regenerated column could be used multiple times with only a slight reduction in these variables, making this novel reactor column an excellent paradigm for high throughput enzymatic generation of proteolytic products from crude samples.
Spot the Difference
The affinity of avidin for biotin has been employed in so many applications that it might seem as if the interaction could hold no surprises. However, a report from Khare and Giometti (p. 584) suggests that even this seemingly routine system may have unappreciated complexities The trouble started during the authors' investigation of the outer membrane proteome of Shewanella. Biotinylation reactions were performed by exposing intact cells to two sulfo-NHS biotin derivatives (which differed only in the length of the spacer arm) either in isolation or as a mixture. After appropriate fractionation steps, the labeled proteins were captured with immobilized avidin in either tetrameric or monomeric form. After elution, the protein preps were run out on a 2-D gel. When the polymeric avidin was used, the whole proved greater than the sum of parts, as novel proteins spots showed up in gels using samples in which the two biotinylation reagents had been combined. Although this trend did not hold true for the mono merit avidin, the experiments performed with this newer form of avidin did show dramatically more spots overall. The authors recommend that researchers considering using the avidin biotin interaction for proteomic analyses consider these factors in se letting the appropriate biotin and avidin reagents.
Finding Binding
Despite the fact that GPCRs have been of intense interest as pharmacologic targets, a significant subset remain orphan receptors, devoid of known function or defined ligands. A means to reconstitute funtional receptors in a defined environment would be of great utility in accelerating understanding of ligand recognition and transmembrane signaling. Nanodiscs, which consist of a nanoscale lipid bilayer core encircled by a membrane scaffold protein, have proven suitable for reconstitution of membrane proteins obtained in purified form or isolated in crude membrane preparations. In work published on p. 601, Leitz et al. describe how Nanodiscs can be used to reconstitute GPCRs that are fully functional. The study focuses on the β2-adrenergic receptor, and provides evidence demonstrating faithful binding of antagonists and agonists. In addition, the β2AR-Nanodisc complex is shown to functionally interact with a soluble G protein, an event that cannot occur when the GPCR is solubilized by detergent treatment. This finding represents the first demonstration that signaling information can be transmitted across a Nanodisc and holds immense promise for drug discovery applications.
Compare and Contrast
The characterization of transcription factor binding sites in groups of related bacterial species aids in the determination of signaling pathways and the identification of important regulatory genes and response elements. Comparisons of species that only recently diverged can yield important information, since these species are more likely to have regulatory pathways and mechanisms in common than more phylogenetically distinct groups. To this end, Conlan et al. have developed a pair of simple Perl script software programs, outlined on p. 578 of this issue, that enable researchers to perform rapid pair wise analysis of orthologous sequences from multiple species. The package generates the results by calling on an external pair wise aligner (Gap, Needle, Water, and Clustal W are supported) and facilitates statistical analysis and display of the output by calculating the mean, median, standard deviation, quartile skewness coefficient, and interquartile range of the percent identity distribution for each pair of aligned sequences. The authors provide results from the comparison of five γ-proteobacterial species, showing that this software can provide a useful and straightforward automated alternative for the generation of data for phylogenetic foot printing studies.
Putting the Hex on HexamersWe have no doubt all been guilty at some stage of taking certain things for granted. This certainly occurs when it comes to following protocols that have been passed down from PI to postdoc for many years. Frequently, however, the so called standard method was decided during the early development of a particular technique and has remained unchanged in the face of concomitant progress in the field. This appears to be the case with reverse transcription PCR. Stangegaard et al. (p. 649) now describe their improvement to this method using longer random pentadecamers (15-mers) compared to the typical random hexamers. The authors tested random primers from 6 up to 21 nucleotides long. Using a standardized template, they demonstrated that 15-mer random primers improved the efficiency of reverse transcription by increasing the yield of cDNA 2 fold over hexamers. No change in the quality or character of the cDNA was seen. They went on to show that pentadecamers could increase the sensitivity of microarray experiments, al lowing for an up to 11-fold increase in the number of genes detected in human transciptome microarrays probed with the pentadecamer generated cDNA. The authors speculate on the reasons for the improvements in transcription using the longer primers, postulating that more favorable hybridization energies may play a role. Although costs might be slightly higher to generate the pentadecamer primers, the payoff in improved cDNA yield and, more importantly, improved representation of the mRNA population is surely worth the extra expense.
