Molecular Biology

DNA ReplicationDNA replication is a fundamental biological process that occurs in all living organisms. Ensuring that it occurs with high fidelity exactly once each time a cell divides is a complex task. Our latest book, DNA Replication, covers all aspects of the replication mechanism and its control. The book was edited by Stephen Bell, Marcel Méchali, and Melvin DePamphilis.

The contributors examine the molecular machinery involved in the assembly of replication origin complexes, the establishment of replication forks, unzipping of the double helix, priming of DNA synthesis, and elongation of daughter strands. Chromatin organization and dynamics, lagging-strand maturation, telomere replication, and mechanisms to handle errors and damage in DNA are also discussed.

“This is a truly exciting field in which to work—the rate of progress of the development of techniques and concepts is remarkable,” write the editors. “As will be apparent from the contents of the book, the full complement of state-of-the-art techniques have been exploited with remarkable effect to tease apart these intricate processes.”

For more information on the book, click here.

Introduction to Protein-DNA InteractionsThe manipulation of DNA by proteins is central to the life of a cell. It is critical for processes ranging from replication and recombination to transcription and the repair of DNA damage. Introduction to Protein-DNA Interactions, written by Gary Stormo, provides an up-to-date and interdisciplinary perspective on protein-DNA interactions, with an emphasis on DNA-binding proteins that control gene expression.

“The regulation of gene expression has fascinated me since my graduate school days,” writes Stormo. “The majority of my efforts have been focused on understanding how networks of transcription factors regulate gene expression and control cell fates and phenotypes. The primary goal of this volume is to provide an introduction to protein-DNA interactions from multiple perspectives.”

Three general types of approaches have been used to study protein-DNA interactions: structural, thermodynamic, and bioinformatic. Stromo describes what we know about protein-DNA interactions from each of these perspectives, and emphasizes how insights from experimental work can be translated into specific computational approaches to create a unified view of the field.

Because of the book’s cross-disciplinary approach, experts working in all fields are certain to learn something new. For more information on the book, click here.

A newborn’s blood spotted onto a Guthrie card. Photo: The New York State Department of Health Newborn Screening Program.

Over the last 50 years, the spotting of newborn’s blood onto filter paper for disease screening, called Guthrie cards, has become so routine that since 2000, more than 90% of newborns in the United States have had Guthrie cards created.  In a study published online in Genome Research (, researchers have shown that epigenetic information stored on archived Guthrie cards provides a retrospective view of the epigenome at birth, a powerful new application for the card that could help understand disease and predict future health.

DNA methylation, an epigenetic chemical modification of DNA, is known to affect gene activity and play a role in normal development, aging, and also in diseases such as heart disease, diabetes, and cancer.  “But are these epigenetic marks involved in causing the disease, or a result of the disease itself?” asked Dr. Vardhman Rakyan of Queen Mary, University of London and co-senior author of the study.  Rakyan explained that this is impossible to know when samples are obtained after onset of the disease.  Guthrie cards, commonly used to collect blood spots from the pricked heel of newborns to screen for diseases such as phenylketonuria, cystic fibrosis, and sickle cells disorders, might offer a snapshot of the epigenome before disease develops.  Many Guthrie cards are stored indefinitely by health authorities around the world, posing a potential wealth of information about the epigenome at birth.

Rakyan and an international group of colleagues purified genomic DNA and analyzed DNA methylomes from Guthrie cards and verified that this archived DNA yields high-quality methylation data when compared to fresh samples.  The researchers then compared the DNA methylation profiles of newborns to the same healthy individuals at the age of three, looking for epigenetic variations detected in the Guthrie card sample that are stable into the early years of life.

“We found similar epigenetic differences between different people both at birth and when they were three years old,” said Rakyan, who added that these differences, already present at birth, are unlikely due solely to inherent genetic differences between the individuals, but also due to environment or random events in utero.  Furthermore, Guthrie card samples could be analyzed for both genetic and epigenetic differences together to view a more complete picture of the genome at birth.

Guthrie card methylomics is a potentially powerful new application for archived blood spots, which could provide a wealth of information about epigenetics and disease, and could give clues about health later in life.  Dr. David Leslie, co-senior author of the study, added that because national health authorities routinely make Guthrie cards available, and with the proper consent obtained from parents and children, “we are talking about an invaluable, and non-renewable, resource for millions of individuals.”

RNA Worlds: From Life's Origins to Diversity in Gene Regulation RNA Worlds is given a positive review by Eugene Koonin in the latest issue of The Quarterly Review of Biology. This “striking volume” is essentially the fourth edition of The RNA World, which is, according to Koonin, “one of the most influential books in biology over the last few decades.”

The first edition of The RNA World was published in 1993; subsequent editions were published in 1999 and 2006, respectively. All discussed the current state of RNA research at the time, and the first two editions, in particular, focused mainly on the domination of RNA during the pre–biotic era. The title of the latest book, RNA Worlds, is “appropriate, significant, and overdue,” says Koonin, because “it has become clear that in many ways we still live in an RNA world.”

Edited by John Atkins, Ray Gesteland, and Tom Cech, RNA Worlds will be a fascinating read for all molecular biologists and biochemists.  For more information on the book, click here.

James Darnell’s recent book is given a wonderful review in the current issue of Cell. “Darnell has succeeded in writing an appealing and cogent account of the rise of RNA molecular biology and its continued centrality in research today,” write the reviewers Kristian Baker and Tim Nilsen.

This is an “excellent book” that describes key historical experiments in a “straightforward and enjoyable way,” say Baker and Nilsen. The “informative figures” and “up-to-date referencing” are a “significant plus.” They conclude that it “should be required reading for graduate students and more senior investigators alike.”

RNA: Life's Indispensable MoleculeWe’ve just published a new book by James Darnell, one of the founding authors of the popular textbook Molecular Cell Biology (W.H. Freeman). In the new book, entitled RNA: Life’s Indispensable Molecule, Darnell provides the first comprehensive account of the history of RNA research from the perspective of his own distinguished, 50-year career at the forefront of the field.

“My aim in writing this book is to provide a supplement in historical form—both to the younger generation of scientists and teachers and through them to incoming students—that describes how we first learned some of the molecular fundamentals of biology,” writes Darnell.

The book will be useful to teachers of undergraduates, as it provides clear descriptions of major developments in the field of RNA research across a historical timeline, beginning over 100 years ago and continuing to the present day. Darnell enthusiastically and eloquently describes the intellectual context in which each question first arose and explains how the key experiments were structured and answers obtained. (more…)

RNA experimentToday’s molecular biologist must be proficient in working with RNA, but experiments with this fragile nucleic acid can be daunting and frustrating.  To help, Don Rio, Manny Ares, Greg Hannon, and Tim Nilsen, all leading authorities in RNA research, have assembled their expertise in a new lab reference – RNA: A Laboratory Manual.

The manual presents a broad range of current techniques used in RNA research, from the most fundamental to the most sophisticated.  It contains detailed step-by-step protocols and extensive tips and troubleshooting information, as well as background information and strategies for approaching any RNA investigation. (more…)

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.

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