Molecular Biology

Cap analysis gene expression (CAGE) is a method used to discover new promoters and for quantifying gene activity, providing data essential for studies of regulatory gene networks. But CAGE requires large amounts of RNA, which are often not obtainable from rare specimens. In the January issue of Cold Spring Harbor Protocols Piero Carninci and colleagues from the RIKEN Yokohama Institute’s Omics Science Center present NanoCAGE: A High-Resolution Technique to Discover and Interrogate Cell Transcriptomes, a method that can capture information from as little as 10 nanograms of total RNA. The protocol describes how to rapidly prepare nanoCAGE libraries which can be sequenced with high sensitivity. As one of January’s featured articles, the protocol is freely available to subscribers and non-subscribers alike.

How do you see something smaller than the wavelength of light itself? Fluorescence microscopy is the most common optical technique used for visualizing cellular functions. The latest techniques allow labeling of specific organelles and proteins with molecular precision. But conventional microscopy cannot resolve objects closer than 200 nanometers at the focal plane. Many subcellular structures and groups of proteins occur on the 10 nanometer scale. A true understanding of cellular physiology requires new superresolution methods.

Of these methods, PALM (Photoactivated Localization Microscopy) provides the highest shown resolution in biological samples (approximately 10 nanometers) and allows for the assessment of individual molecules. In the December issue of Cold Spring Harbor Protocols, Oregon Health Science University’s Haining Zhong presents Photoactivated Localization Microscopy (PALM): An Optical Technique for Achieving ~10-nm Resolution. The article provides an overview of the basic principles of PALM, its implementation and the potential applications in neuroscience.

Blood feeding mosquitoes transmit many of the world’s deadliest diseases, which are resurgent in developing countries and pose threats for epidemic outbreaks in developed countries. Recent mosquito genome projects have stimulated interest in the potential for disease control through the genetic manipulation of vector insects. To accomplish this, vector insects must be established as laboratory model organisms, allowing for a better understanding of their biology, and in particular, the genes that regulate their development. Aedes aegypti is a vector mosquito of great medical importance because it is responsible for the transmission of dengue fever and yellow fever. In the October issue of Cold Spring Harbor Protocols, Molly Duman-Scheel and colleagues present an overview of the background, husbandry, and potential uses of Ae. aegypti as a model species. Protocols are provided for culturing and egg collection, fixation and tissue preparation, whole mount in situ hybridization, immunohistochemical analysis and RNA interference in Ae. aegypti. This methodology, much of which is applicable to other mosquito species, is useful to both the comparative development and vector research communities.

This article series marks the latest entrant in Cold Spring Harbor Protocols’ long-running series on Emerging Model Organisms.

Post-translational modifications of histones play an important role in regulating chromatin dynamics and function. One such modification, methylation, is involved in the regulation of the epigenetic program of a cell, determining chromatin structure, and regulating transcription. Methylation of histones occurs on both lysine and arginine residues, and until recently, was thought to be an irreversible process. The recent discovery of histone demethylases revealed that histone methylation is more dynamic than previously recognized. The October issue of Cold Spring Harbor Protocols features a set of methods from Keiichi Nakayama and colleagues from Kyushu University for investigating demethylase activity. The protocol, In Vitro Histone Demethylase Assay, describes two different in vitro histone demethylase enzyme reactions and three different methods for measuring histone demethylase activity. These methods can be applied to measuring histone demethylase activity in tissues and cell lysates, identification of novel histone demethylases, and screening for inhibitors of histone demethylases. As one of our featured articles, the protocol is freely available to subscribers and nonsubscribers alike.

A cell devotes a significant amount of effort to maintaining the stability of its genome, preventing the sorts of chromosomal rearrangements characteristic of many cancers. Assays that measure the rate of gross chromosomal rearrangements (GCRs) are needed in order to understand the individual genes and the different pathways that suppress genomic instability. In the September issue of Cold Spring Harbor Protocols, Richard Kolodner and colleagues from the University of California, San Diego’s Ludwig Institute for Cancer Research present Determination of Gross Chromosomal Rearrangement Rates, a genetic assay to quantitatively measure the rate at which GCRs occur in yeast cells. The assay measures the rate of simultaneous inactivation of two markers placed on a nonessential end of a yeast chromosome. This simple protocol for determining GCR mutation rates in a variety of genetic backgrounds coupled with a diversity of modified GCR assays has provided tremendous insight into the large numbers of pathways that suppress genomic instability in yeast and appear to be relevant to cancer suppression pathways in humans. As one of September’s featured articles, the full text protocol is freely available to subscribers and nonsubscribers alike.

Large segments of DNA can vary in copy number between individuals. Such copy number variations (CNVs) contribute greatly to genetic diversity and are also thought to be associated with susceptibility or resistance to some diseases, including cancer. Simple Copy Number Determination with Reference Query Pyrosequencing (RQPS), featured in the September issue of Cold Spring Harbor Protocols, provides an assay for determining the copy number of any allele in the genome. The method, from Raphael Kopan and colleagues at Washington University, takes advantage of the fact that pyrosequencing can accurately measure the ratio of DNA fragments in a mixture that differ by a single nucleotide. A reference allele with a known copy number and a query allele with an unknown copy number are engineered with single nucleotide variations, and the ratio seen between these probes and genomic DNA reflects the copy number. RQPS can be used to measure copy number of any transgene, differentiate homozygotes from heterozygotes, detect the CNV of endogenous genes, and screen embryonic stem cells targeted with bacterial artificial chromosome (BAC) vectors. RQPS is rapid, inexpensive, sensitive, and adaptable to high-throughput approaches. As one of our featured articles, the protocol is freely available to subscribers and non-subscribers alike.

Improvements in automation and acquisition time have made the microscope a viable platform for performing hundreds of concurrent parallel experiments. Using these sorts of tools, it is now possible to run high-throughput screens for protein function and interaction in living cells, examining dynamic cellular processes to distinguish between primary and secondary phenotypes, and to study the phenotype kinetics. In the August issue of Cold Spring Harbor Protocols, Jan Ellenberg and colleagues from the EMBL present High-Throughput Microscopy Using Live Mammalian Cells, an overview of how to screen live cells using imaging technologies. The article examines each aspect of the general screening process and considers specific examples in the processing of time-lapse experiments. The techniques discussed are based on the use of cultured mammalian cells, but the concepts are easily transferred to cultured cells from other species like Drosophila and small organisms such as C. elegans.

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