Advances in microscope instrumentation have allowed for the acquisition of increasingly large and information-rich digital image files. Although this wealth of data is good, these experiments have a dark side since most automated image analysis programs lack the versatility to define different biological objects. This means some images may have to be analyzed or corrected manually, a daunting task for even the most diligent biologist. Lamprecht et al. are trying to address this problem through the image analysis software package called CellProfiler™. CellProfiler consists of independent modules with the capacity to process images, correct for illumination problems, and identify, measure, and record a wide array of biological objects by shape, size, texture, color-intensity, and number of neighbors. These independent modules can be configured together into a “pipeline” for any experiment. The authors demonstrate the utility of CellProfiler through five different applications of the program: (i) assessing yeast colony morphology and number; (ii) annotation and analysis of cell-microarrays using grids placed on the image; (iii) quantification of mouse tumor size and number; (iv) analysis of cell patterning during wound healing assays; and (v) measurements of tissue topology. Although described using these five applications, CellProfiler has the ability to address many more biological questions simply by changing existing modules or creating new ones to design new pipelines. CellProfiler is a welcome entry into the image analysis arena, providing biologists a new suite of tools to improve the automated analysis of microscopy data. -Page 71
“A Reporter for All Seasons”
Molecular biologists use reporter gene detection systems every day. From the IacZ encoded enzyme β-galactosidase and its substrate X-gal for routine molecular cloning to green fluorescent protein (GFP) for in vivo imaging, reporter genes are essential to life science research. However, each reporter system has drawbacks, from unsuitability when there is a need to work with nonfluorescent substrates (IacZ with X-gal) to low sensitivity (GFP), no single reporter system seems appropriate for all applications. Qureshi makes an argument in his mini-review that TEM-1 β-lactamase (BLA) and its fluorescent substrates might be the one reporter system that overcomes these drawbacks and provides a robust system for use in most applications. Fluorescent BLA substrates CCF2/CCF4-AM are able to traverse cell membranes easily with minimal trauma to the cells and become trapped inside the cell due to negative charge. These substrates can be detected by FRET with emission of green light (λ = 530 nm) prior to cleavage by β-lactamase; upon cleavage, the substrates separate into two fluorophores, leading to loss of FRET and emission of blue light (λ = 460 nm). To validate the usefulness of the BLA system, the author describes experiments where a recombinant glucocorticoid receptor (GR) was engineered in cells to induce BLA expression upon activation. These cells could be subsequently loaded with the BLA substrate CCF4-AM and activation monitored with FACS, providing a high-throughput assay for activators of GR. β-lactamase just might be a “reporter for all seasons.” -Page 91
As technology advances and researchers increasingly rely on archival tissue to apply new assays to old samples, whole-genome amplification (WGA) has been widely used to generate large amounts of DNA from miniscule amounts of available tissue. One of the most popular approaches to WGA is multiple displacement amplification (MDA) due to its high genotype call concordance between amplified and nonamplified templates and between MDA-amplified and non- MDA-amplified templates. In this issue, Corneveaux et al. show that MDA may generate chromosomal copy number artifacts. They conducted chromosomal copy number analysis on MDA-amplified DNA of 532 individuals and compared these to a control sample of pooled DNA from 100 healthy donors using the Affymetrix 10K GeneChip® Human Mapping Array—a panel of approximately 10,000 SNPs. While the average copy number for SNPS in the control sample was 2; in the MDA-amplified group, approximately 1 in every 7 SNPs showed copy number change. The authors caution that while MDA treatment of DNA may be the method of choice for genotyping purposes, caution should be exercised when drawing conclusions about copy number from these samples. They also advise that untreated tissue should be retained from biopsies until more reliable approaches to determining copy number have been developed. -Page 77
Reducing PCR Bias in Methylation AnalysisMany analyses of DNA methylation commence with bisulfite treatment of genomic DNA, which converts unmethylated cytosines to uracils, leaving methylated cytosines unchanged. Bisulfite-induced sequence changes can be characterized, following PCR, by a number of approaches, including sequencing and restriction enzyme analysis. It has been reported that PCR amplification of bisulfite-treated DNA results in bias in the form of preferred amplification of unmethylated sequences, presumably as a result of secondary structure effects on amplification of the more GC-rich methylated sequences. Shen et al. address this issue by optimizing PCR annealing temperature and analyzing sequence changes using pyrosequencing. They find that, independent of the primer system used, higher annealing temperatures significantly improve the efficiency of amplification of unmethylated DNA sequences. Implementation of this approach may serve to advance the field of epigenetics by significantly improving the accuracy of DNA methylation analysis. -Page 48
Carrier Proteins Help Define Phosphatases
Protein biochemists know how difficult it can be to identify enzyme substrates. In the world of protein phosphatases, defining the specificity of a particular phosphatase can be greatly hampered by the time-consuming and technically challenging methods required to generate protein substrates. Studies of protein phosphatases have traditionally relied on either the use of these difficult-to-generate phosphoproteins or quicker to obtain, but less effective, synthetic phosphopeptides as substrates. Kochinyan et al. provide a promising fix to this dilemma by using an intein-mediated protein ligation technique to fuse specific phosphopeptides to carrier proteins resulting in ligated phosophoproteins (LPP). They describe the generation of LPP substrates that contain phosotayrosine, phosphoserine, or phosphothreonine residues. Using these three LPP substrates, the authors determined the substrate specificities of three different protein phosphatases and found a 240-fold increase in sensitivity in dot blot analysis when compared to the same assay using phosphopeptides alone. This technique has the potential to allow rapid identification of different phosphatase specificities, without the need for laborious phosphoprotein production or the use of less than optimal phosphopeptides. -Page 63
Protein Design
Design of proteins having multiple identical domains facilitates analyses of the effect of domain copy number on structural and functional properties of proteins, such as folding, activity, and binding affinity. However use of traditional approaches to gene synthesis for this application is not without obstacles. Achmüller et al. present a new method for generating recombinant proteins having multiple identical domains and apply it to streptococcal protein G domain B1 (SpG-B1). The domain of interest was synthesized using a traditional gene assembly method, followed by linking of single domains by PCR using forward and reverse link-primers containing the reverse complementary sequence of the 3′ and 5′ ends of the amplified fragment, respectively. A second PCR using forward and reverse adaptor-primers, containing anchor and restriction sites, permitted elimination of the 5′ and 3′ ends produced in the first PCR and cloning of the desired number of domains. After electrophoresis in agarose, fragments containing the desired number of domains were extracted and amplified for subsequent cloning. The authors were successful in producing gene constructs for SpG-B1 containing one, two, and three copies of the domain. This highly specialized approach will be a valuable addition to the armamentarium of protein design tools. -Page 43
