Age Discrimination

Now that microarrays for measuring steady-state levels of gene expression have become routine, increasing interest has turned to quantitatively assessing nascent transcription. Previously, looking at nascent mRNAs meant performing nuclear run-on assays, which offer quite limited throughput. Microarray-based approaches circumvent that limitation, but to date there has not been a published method that offers a convenient, optimized means to label, purify, and quantitate nascent mRNAs using standard reagents. Writing in DNA Research, Ohtsu et al., fill that gap by describing in detail their method for performing transcriptional analysis of cell cycle regulation factors in mouse mammary carcinoma cells by using bromouridine labeling and immunoprecipitation with anti-BrdU antibody. The authors faced two significant challenges. First, the labeled nascent RNA is a small subset of total RNA. Second, the overall amount of newly synthesized RNA is extremely limited. They found that the addition of excess Escherichia coli rRNAs to the immunoprecipitation reaction as a blocking agent significantly improved the specific recovery of labeled RNA. Similarly, simple modifications to a commercial amplification kit produced cDNA with little bias as assessed by quantitative reverse transcription–PCR validation of microarray results. Oligo microarray analysis of nascent versus steady-state transcripts in the cell cycle of synchronized and asynchronous cells implicated several genes in cell cycle control or response. Beyond this application, the method should also be applicable to any situation in which it is necessary to detect gene expression changes with high sensitivity.

Ohtsu et al. 2008. Novel DNA microarray system for analysis of nascent mRNAs. DNA Res. 152:41-251.

Lather Up

Membrane proteomics is nearly impossible without the use of detergents. Although there are alternatives that rely upon solubilization solutions such as 60% methanol or 90% formic acid, detergents are generally better at this task and so are preferred for proteomic analysis. Unfortunately, detergents obscure peptide analysis by mass spectrometry. Though approaches like ultrafiltration, density gradient centrifugation, and dialysis have been used for detergent removal, such methods are not widely used prior to mass spectrometry because they tend to leave residual detergent. For that reason, gel electrophoresis and in-gel digestion have become the standard pre-mass spectrometry (MS) separation approach. Now, a paper from Nagaraj et al., in the Journal of Proteome Research offers a gel-free means of detergent removal. The authors generated crude membrane preparations from mouse brain using SDS, Triton X-100, or CHAPS. The solubilized membrane proteins were then loaded on a desalting column pre-equilibrated with 8 M urea. This chaotro-pic agent dissociates the micelles while retaining the membrane proteins in solution. Unlike the proteins, which elute in the void volume, the detergent enters the gel filtration matrix and so is separated from the protein fractions. After digestion and liquid chroma-tography (LC)-MS/MS, almost 80% of the identified proteins were from membrane. Significantly, over two times as many proteins were identified after sample preparation by using the urea-equilibrated column compared with traditional in-gel detergent removal. Proteins were recovered from a variety of membranes, including mitochondrial, plasma, nuclear, and ER. Although there were no consistent properties of the proteins that were picked up by the new method, the technique did seem to be better for recovery of large (>1,500 amino acid) proteins. The method proved equally adept when applied to samples from liver, spleen, eye, and muscle. Besides the obvious advantages in proteome coverage, Nagaraj et al.'s, method does not require extensive preanalysis fractionation. Compared with other published membrane proteomics efforts, the detergent-based, gel-free method is likely to be attractive for high-throughput experiments or applications in which sample sizes are too small for straightforward organelle purification.

Nagaraj et al. Detergent-based but gel-free method allows identification of several hundred membrane proteins in single LC-MS runs. J. Proteome Res. [Epub ahed of print, October 10, 2008; doi: 10.1021/pr800412j].

Saturation Coverage

For most people, watching their saturated fat means skipping a slice of cheesecake after dinner, but for Rinia et al., it entails detailed chemical characterization of fatty acids in lipid droplets. As unappealing as they sound, lipid droplets are important organelles which function in energy storage, biosynthesis of membrane lipids and steroid hormones, and regulation of cholesterol levels. Although most common in adipocytes, lipid droplets can form in any cell, as seen in the visceral fat deposition implicated in type 2 diabetes. Previous studies have suggested that lipid droplets are quite heterogeneous, but fuller characterization has been limited by the lack of methods to measure the chemical composition and physical state of individual lipid droplets. As described in a recent article in the Biophysical Journal, one existing method relies upon coherent anti-Stokes Raman scattering (CARS) microscopy, a technique that uses the vibrational properties of C-H and C = C bonds to infer the amount of acyl chain order and the degree of fatty acid unsaturation, respectively. The advance described in this paper is the use of multiplex CARS and quantitative analysis by means of the maximum entropy method. The result is that measurements of the specific properties of fatty acids can be made with submicron spatial resolution, allowing a quantitative picture of lipid properties within a small localized area, such as a cell or within an individual lipid droplet. As evidence, the authors offer results from their study of mouse adipocytes incubated with media containing defined mixtures of palmitic acid (fully saturated) and linolenic acid (with three double bonds). Subsequent imaging revealed that the technique was able to pick up variations in lipid composition between and even within lipid droplets, including providing a three-dimensional map of acyl order and unsaturation. Such information should provide a fuller picture of formation and growth of lipid droplets. Extensions to the technique, including use of living cells and the possibility of simultaneous fluorescence imaging in order to track lipid droplet-associated proteins, promise to trigger more interest in lipid droplets and accelerate understanding of the complexities of these underappreciated organelles.

Rinia et al. Quantitative label-free imaging of lipid composition and packing of individual cellular lipid droplets using multiplex CARS microscopy. Biophys. J. [Epub ahead of print, August 8, 2008; doi:10.1529/biophysj.108.137737].

Nuclear Annihilation

One of the best ways to figure out the function of a nuclear factor is to take it out of commission and watch the consequences. In yeast, conditional knockouts have typically been realized by turning up the heat on temperature-sensitive mutants. However, Haruki et al., weren't having much success with that popular technique when they tried to apply it to nuclear pore components, given that the phe-notypes seen may have resulted from nonspecific stress responses to the temperature increase. As an alternative, the authors developed a sequestration method that they describe in a recent issue of Molecular Cell. The approach rests on the rapamycin-dependent affinity between the FK506 binding protein (FKBP12) and the FKBP12-rapamycin-binding (FRB) domain of mTOR. The yeast large ribosomal subunit was tagged with FKBP12, and the target factor to be depleted was fused to FRB. Ribosomal proteins are transported to the nucleus, assembled with rRNA into ribosomal subunits, and then exported to dwell in the cytoplasm. This life cycle makes ribosomal proteins ideal “anchors” to bind and trap nuclear factors in the cytoplasm. In tests with sequestered TATA-binding protein, transcription was strongly inhibited in the presence of rapamycin, an effect which is not due to general toxicity because expression of untagged TBP reverses the pheno-type. All told, the authors successfully tested their anchor in over 40 different strains containing tagged target proteins. This suggests that the “anchor-away” strategy will be a robust technique appropriate for teasing apart the function of nuclear factors in the yeast experimental system.

Haruki et al. 2008. The anchor-away technique: rapid, conditional establishment of yeast mutant phenotypes. Molecular Cell 31:925-932.