Ch-ch-ch-changes: The Evolution of “Mutation”

Mutations are central to biology—they explain diversity in life forms, provide fuel for evolution, and determine one’s susceptibility to certain diseases.  But scientists have not always understood mutations as we do now—as molecular alterations in DNA.

“Mutation, of course, involves change,” writes Elof Axel Carlson. “But our understanding of that change is influenced by the time we live in.”  In his latest book, Carlson explores the history—the people, science, and ideas—behind the concept of mutation.

Carlson describes how the idea of mutation has changed considerably from the pre-Mendelian concepts of Darwin’s generation over 150 years ago. Darwin viewed “fluctuating variations” as the raw material on which evolution acted.

 

Collection of Genetics Interviews is “Inspirational”

Speaking of Genetics, Jane Gitschier’s collection of interviews with prominent scientists and non-scientists involved in genetics, is reviewed in the current issue of CHOICE.  In the review, Randall Harris (William Carey University) says the interviews “give the reader the feeling of sitting in on an evening gathering among friends.”

Researchers convey the exhilarating moments of discovery, as well as the hard work, serendipity, joy, and frustration involved. Harris says the book “will prove inspirational to scientists at all stages of their careers.”

 

The Biology of the Human Genome

There’s much excitement here at CSHL in anticipation of the popular annual Biology of Genomes meeting, which begins tonight. For the next 6 days, scientists from around the world will assemble on campus to discuss the latest advances in genome research as they relate to evolution, biology, and disease in a variety of organisms—including humans.

This year marks the 10^th anniversary of the publication of the draft human genome sequence. What has it told us about human biology? Do we know the function of every gene in the human genome?

To address these questions, Stewart Scherer has spent substantial time and effort compiling information from the scientific literature about human genes and their biological functions.  The result is a new online resource, Guide to the Human Genome, that puts the genes of the human genome in their biological context. The Guide, available at www.humangenomeguide.org, provides extensive up-to-date information about human genes in an easily accessible format.

 

NanoCAGE

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.

 

Disease Vector Mosquitoes in the Laboratory

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.

 

Histone Demethylase

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.

 

Chromosomal Rearrangement

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.

 

Determining Copy Number Variation (CNV)

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.

 

High-throughput Screening of Living Cells

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.

 

Zinc Finger Nuclease Deletions

Zinc finger nucleases (ZFNs) are artificial restriction enzymes made by fusing an engineered zinc finger DNA-binding domain to the DNA cleavage domain of a restriction enzyme. ZFNs can be used to generate targeted genomic deletions of large segments of DNA in a wide variety of cell types and organisms. In the August issue of Cold Spring Harbor Protocols, Jin-Soo Kim and colleagues present Analysis of Targeted Chromosomal Deletions Induced by Zinc Finger Nucleases, a detailed protocol for the detection and analysis of large genomic deletions in cultured cells introduced by the expression of ZFNs. The method described allows researchers to detect and estimate the frequency of ZFN-induced genomic deletions by simple PCR-based methods. As one of our featured articles, the protocol is freely available to subscribers and non-subscribers alike.