Interactions between transcription factors and their target DNA mediate a multitude of biological processes. The chromatin immunoprecipitation (ChIP) assay is a tool for isolating and identifying the gene sequences bound by a particular protein of interest in living cells and can offer early insight into the mechanics of important protein-DNA interactions. In their review, Das et al. (p. 961) present a detailed discussion of the techniques that constitute the ChIP assay—methods for chromatin fixation and fragmentation and immunoprecipitation of protein with bound DNA—as well as the advantages and limitations of each approach. The authors also discuss hybrid methods including ChIP-chip (ChIP in combination with whole-genome microarray), ChlP-CpG (ChIP in combination with CpG microarray), and cloning and sequencing of ChIP-isolated DNA. This review will serve a wide audience as a useful primer on the pros and cons of ChIP.Dance of the Dictyostelium
Like square dancers dosey-doeing in time to a fiddle, Dictyostelium discoideum amoebae aggregate in rhythmic movements coordinated by cAMP secretion. This process, by which single amoebae form a multicellular organism, is an important model system for studying differentiation. Researchers have traditionally measured aggregation photometrically by placing Dictyostelium suspensions in a cuvette and following light scattering oscillations over time. While this system works well, measuring one sample at a time is not well suited to the task of untangling a complex oscillatory network involving multiple parameters. To address this deficiency, Lusche et al. (p. 970) describe a multichannel apparatus capable of parallel monitoring of light scattering in up to eight Dictyostelium cultures. This innovation allows experiments involving side-by-side comparisons of mutant and wild-type cell suspensions as well as synchronized screening of the effects of agents that interfere with signal transduction. The authors validate their system by showing expected results with both a previously characterized mutant and a well understood chemical intervention. With a computer-controlled setup allowing straightforward determination of oscillation parameters like phase length and amplitude, researchers using this system might well wish to join their Dictyostelium suspensions in a dance of joy.Hot Start RD-PCR
By its nature, PCR suffers from inherent variability caused by sensitivity to the amount of starting template. This is particularly problematic when attempting to perform quantitative experiments. While using a new robust dosage PCR (RD-PCR) technique to detect large heterozygous deletions and duplications, Shi et al. (p. 934) were able to show that pre-incubation of DNA at 90°C for 10 min in 2× TE buffer could decrease variability by several fold. This treatment, which worked as well as an overnight incubation with Proteinase K, is hypothesized to disrupt the interaction of high-affinity DNA binding proteins that may negatively affect amplification. Although further supporting work is necessary, this advance has the potential to significantly improve the reliability and reproducibility of many PCR-related methodologies.Eyeing Islets
Although a sedentary lifestyle can contribute to the risk of developing type 2 diabetes, the insulin secretion pathway is anything but listless. Upon metabolism of glucose within the β-cells of the islets of Langerhans, a series of events begins that causes an increase in the intracellular Ca2+ concentration. Intracellular Ca2+, in turn, spurs exocytosis of vesicles containing insulin:Zn2+ complexes. Clearly, this dynamic and complicated process could benefit from real-time analytical techniques. While a variety of imaging, ampero-metric, and immunoassay techniques have been described for studying insulin signaling, these methods are not well equipped for simultaneously monitoring intracellular Ca2+ concentration and insulin:Zn2+ secretion on a fast time scale. Qian et al. (p. 922) draw upon their previous experience with a fluorogenic Zn2+-specific indicator and show that it can be used with the Ca2+ marker Fura Red to simultaneously view these cations in islet cell clusters and whole islets. In particular, the strategy is adept at following synchronous oscillations of Zn2+ secretion and intracellular Ca2+ that occur on a fast (tens of seconds) time scale. This behavior suggests that the Zn2+ is in fact a useful surrogate of insulin release. Moreover, the spatial and temporal resolution possible with this technique should make it applicable for screening drugs for effects on β-cell secretion and further discerning the intracellular signaling that defines islet function.
A New Enzyme on the Block
Originally patented for more industrial uses, namely the hydrolysis of plant material into fermentable sugars, α-L-arabinofuranosidase has been reconceived as a tool for the molecular biologist. Tsuchida et al. (p. 896) demonstrate that this enzyme (encoded by the abfA gene), when used in conjunction with a synthetic substrate, Z-ara, can be detected and visualized in a manner identical to the ubiquitous LacZ system. They have further shown that cotransfection of abfA and LacZ—the latter linked to a nuclear localization signal—into NIH 3T3 cells yielded clearly different and distinguishable staining patterns: a blue cytoplasm from the Z-ara substrate and a red-purple nucleus resulting from hydrolysis of Magenta-Gal, an X-gal analog. No cross-reactivity of substrates was seen. The absence of α-L-arabinofuranosidase or its natural substrates in mammalian cells creates a system with very low background, allowing it potentially to replace LacZ in assays where high levels of endogenous β-galactosidase are present, as well as making it an excellent companion to the LacZ system for dual detection of gene expression in both cultured cells and transgenic animals.
Real-Time RT-PCR Gets SASsy
Real-time reverse transcription PCR (RT-PCR) has become a popular method for studying the expression of low abundance genes. For results to have the required accuracy and reliability, however, a reproducible methodology coupled with a suitable mathematical model must be found. Furthermore, the analysis of independently obtained samples, as opposed to replicate samples, requires that additional confounding variables be taken into account. Through analysis of raw real-time RT-PCR data using the commercially available SAS statistical package, Cook et al. (p. 990) have developed a means to estimate the relative gene expression ratio between two populations and assign confidence intervals to this estimate. This process means that results from two separate experiments (performed, for example, on different days) can be more confidently compared. Real-time RT-PCR methodology can thus be more generally applicable and, in the opinion of the authors, as useful as microarrays for the simultaneous analysis of a moderate number of genes.MaGIC Marker
These days, discovery of disease-associated genes is anything but child's play. The low-hanging fruit of single gene disorders have already been plucked, leaving the daunting problem of deciphering complex diseases with multiple genetic and environmental triggers. Fortunately, there are now several extensive databases of polymorphic markers, including microsatellite and single nucleotide polymorphism (SNP) resources. The downside of such a wealth of data is, however, that current computational power is not yet up to the task of performing a whole genome screen with all available markers. Obviously, therefore, the success of a complex disease association study depends on the suitability of the marker set selected. To assist in this important step, Simpson et al. (p. 996) have created MaGIC (Marker Gene Interspacing and Correlation), a program that generates lists of markers correlated with other genomic features. The program works on the premise that a restricted subset of markers can hold the same information as the full set, provided that the selected markers are confined to genomic features of interest (e.g., exons). This strategy represents a useful complement to the International HapMap project, which is still in the process of converting genotype data to a genome-wide representation of linkage disequilibrium; moreover, these data are being incorporated into MaGIC in the form of marker spacing measured by linkage disequilibrium units (LDUs). Users can select markers at any density and on the basis of such criteria as validation status, heterozygosity, or inclusion in the HapMap project. The program also permits users to import custom genome data in the form of in-house annotations, marker lists, or data from related species. In short, MaGIC represents an attractive means to balance workload, cost, and power in genetic association studies, whether the goal is genome-wide analysis of feature-rich regions or fine-mapping studies for the final stages of a gene-hunting project. The program and source code are freely available at http://cogent.iop.kcl.ac.uk/MaGIC.cogx .