Pillars of Truth

Small vibrations that occur during microscopy can often lead to big problems with the visualization of single molecules or the interpretation of subcellular processes. Often these small vibrations come from the microscope itself, caused by instrument parts such as a motorized stage or an attached CCD camera. This can become especially problematic when the experimental goal is to track the nanoscale movement of cellular organelles such as vesicles. In this issue, Dangaria et al. provide an inexpensive fix to this problem by microfabricating fiduciary posts attached to coverslips and using these posts to determine amounts of low frequency positional drift. The posts, 10 µm high by 3 µm in diameter, allow tracking of coverslip motion, thus providing a means to subtract coverslip motion or drift from the movement of cellular organelles. The authors examined the amount of drift due to vibrations from a motorized stage, finding an average drift velocity of 0.3 µm/s. This coverslip drift could then be accounted for by simply subtracting the corresponding movement of a fiduciary post. Using this procedure, the authors were able to classify vesicle movements into categories such as diffusive, subdiffusive, and directed motion, allowing discrimination between Brownian motion and motor-driven transport of molecules in cells. An additional benefit of these posts is the potential to follow the motion of vesicles in three dimensions by relating movement to the fiduciary post. These new microfabricated posts provide an excellent, inexpensive means to improve tracking at the nanometer level and should become a widely adopted microscopy tool. -Page 437

I Smell a Rat…Reference Gene

Real-time reverse transcription PCR (RT-PCR) is an important tool for gene expression profiling. A major problem associated with this technique is controlling the levels of variability so that meaningful data regarding mRNA expression levels can be obtained. The variability of RT-PCR can come from many sources, including sample variation and variation in the efficiency of the enzymes used in the PCR. To minimize this variability, it is critical to utilize a reference gene to normalize the results; however, even choosing a good reference gene can be difficult. Cai et al. examined several possible sources of variation associated with RT-PCR in rat tissues and determined suitable reference genes for various tissue types. The authors evaluated a panel of 48 genes in 11 different tissue types and determined the variability in gene expression based on cycle threshold measurements. Using a mathematical model to determine the sources of variability, it was shown that tissue type accounts for the greatest variability in gene expression with animal and replicate number accounting for considerably less variation. Several genes were identified as potentially good reference genes for RT-PCR experiments involving different rat tissues, some even better than traditionally used housekeeping genes such as β-actin and 18s rRNA. The authors have provided a set of validated reference control genes for performing RT-PCR experiments in the rat, which should be very useful to scientists given the role the rat currently has in preclinical research. -Page 503

Flatbed Scanners for Densitometry

Use of flatbed scanners as a cost-effective tool for densitometry has been explored in the past, but wide-spread use of the scanners for this purpose has not materialized. Tan et al. revisit this issue, focusing on the influence of light spectrum in densitometry. The authors use proteins separated in a polyacrylamide gel and stained with Coomassie™ Blue for their analysis. By a combination of numerical modeling and experimentation, they demonstrate that the spectrum of light has pronounced effects on the dynamic range of the scanner, with a higher scale resulting in improved sensitivity. They achieve the highest optical density range with a red LED light. Noting that flatbed scanners operate on the principle of reflectance—with the light source and detector located on the same side of the sample—the authors adapt their scanner using a minor adjustment to permit the scanner to operate under the principle of transmission. This entails use of the original light source to perform initialization; this light is then switched off to permit use of any secondary light source for scanning. Under these conditions, the authors show that the adapted scanner is capable of achieving a level of linearity and sensitivity comparable to a widely used commercial densitometer. -Page 474

Biologists Get More PC

Scientists interested in studying cell membrane associated lipids often encounter difficulty due to the nonspecific extraction methods used to isolate these lipids. New methods that could easily and rapidly isolate specific amphipathic lipids would be extremely useful for the biologists studying membrane biology or diseases associated with problems in lipid metabolism. Vassar et al. describe a new method that specifically isolates phosphatidylcholine (PC), a major component of the eukaryotic cell membrane. Traditionally, PC has been isolated in a two-step process involving the isolation of total lipids followed by a purification step, usually by either thin layer chromatography or high-performance liquid chromatography, to obtain pure PC. This procedure is time-consuming, requiring from 1 to 2 days to fully complete. Here, the authors have devised a simple, elegant system where a single extraction using a 9:1 hexanes to isopropanol mixture isolates pure PC in only 30 min. The authors compared the new method with two traditional extraction methods demonstrating the use of 9:1 hexanes to isopropanol is as effective at isolating PC, although requiring significantly less time. This new method to extract pure PC will certainly be useful for membrane biologists, as well as other scientists interested in purifying and studying these lipids. -Page 442

Unmarked Gene Modification in Streptococcus mutans

Genetic analysis of Streptoccus mutans and other streptococci has advanced more slowly than that of other microbes as a result of a paucity of appropriate genetic tools. S. mutans is a low G+C Gram positive bacterium capable of achieving moderate levels of competence relative to highly competent streptococci such as S. pneumoniae. In this issue, Biswas et al. demonstrate that S. mutans is cotransformable and describe an approach to achieve unmarked gene selection in this species. The authors use a circular thermosensitive plasmid that has a broad host range and which bears an erythromycin resistance cassette for selection and a linear DNA molecule to target the gene of interest. Following selection and confirmation by PCR that the desired unmarked recombination event had taken place, the selection plasmid is cured from the cells by growth at a temperature nonpermissive for selection plasmid replication. The approach was successful in introducing insertions and deletions and will be applicable to use in other transformable streptococci, as well as toward furthering genetic analyses of S. mutans. -Page 487

Insertional Mutagenesis in Quasi-Haploid Cells

The creation of novel phenotypes for analysis of gene function can be accomplished in vivo using germline mutagenesis or in vivo using cultured cells and chemical or insertional mutagenesis. Both approaches have advantages and limitations. In the cell culture model, the diploid nature of cells is a major obstacle to generation of a dominant phenotype because, in general, only a single copy of each gene is modified by mutagenesis. In this issue, Kim et al. describe a novel method to achieve high rates of mutation in cultured cells for functional genomics studies. They demonstrate that exposure of cells to 6–8 rounds of chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) results in a mutation rate of 10⁻¹ and produces functionally haploid cells. Following chemical mutagenesis, cells were selected for resistance to lethal toxin-induced cell death and then further mutagenized using a retroviral insertion vector. Insertional mutagenesis was followed by selection for antibiotic resistance and again for resistance to lethal toxin. 3′-RACE and sequence analysis were used to identify the genes disrupted by insertional mutagenesis, and those genes were then silenced in wild-type cells using RNAi to confirm their participation in conferring lethal toxin resistance. While this system has limitations, the approach can be applied to a variety of cell types and experimental situations, and the concept can be applied to germline mutagenesis. -Page 493