During the last few years, new discoveries in the RNA field have opened up a wealth of opportunities to specifically target mRNA for functional analysis and target validation. Contained in this volume of Methods in Molecular Biology are methods useful for the design and application of ribozymes, DNAzymes, and small interfering RNAs (siRNAs). In addition, a number of illustrations aiming to facilitate the und- standing and application of each method is included. Like any new field, ribozymes and siRNA research is rapidly evolving, and so too are the methods used to study, select, express, and...

The Right Book at the Right Time The poxviruses comprise a family of complex DNA viruses that replicate in the cytoplasm of vertebrate or invertebrate cells. Of the eight recognized g- era of vertebrate poxviruses, those belonging to the orthopoxvirus genus have been most intensively studied. This group includes variola virus, the agent of smallpox, as well as cowpox virus and vaccinia virus. Jenner s original sma- pox vaccine, described in 1798, consisted of live cowpox virus, but vaccinia virus later replaced it (1). There has been speculation as to the origin of v- cinia virus; the most...

Protein Arrays: Methods and Protocols is an introduction to protein array technology and its application to the multiplexed detection of proteins. Although protein array technology has some roots in gene array technology, it can only be described as a distant relative. Unlike DNA, with its established rules of base pairing, and therefore predictable biochemical behavior, proteins are rich with diversity. Proteins can be large or small, compact or extended, basic or acidic, hydrophobic or hydrophilic, and so on. Just as importantly, their behavior is determined by the environment in which they...

The study of germ cells has undergone enormous advances in recent years and has entered into an explosive phase of new discoveries with the introd- tion of transgenic technologies and nuclear cloning. Basic knowledge and te- niques developed for lower vertebrate and invertebrate systems have facilitated the study of higher vertebrates, including humans. Many experiments that have first been performed on lower vertebrates provided the tools and strategies that could later be applied to other less readily available mammalian systems. The discovery of centrosomes in ascidians and sea urchin eggs...

In the literature, several terms are used synonymously to name the topic of this book: chem-, chemi-, or chemo-informatics. A widely recognized de- nition of this discipline is the one by Frank Brown from 1998 (1) who defined chemoinformatics as the combination of all the information resources that a scientist needs to optimize the properties of a ligand to become a drug. In Brown s definition, two aspects play a fundamentally important role: de- sion support by computational means and drug discovery, which distinguishes it from the term chemical informatics that was introduced at least ten...

Directed evolution comprises two distinct steps that are typically applied in an iterative fashion: (1) generating molecular diversity and (2) finding among the ensemble of mutant sequences those proteins that perform the desired fu- tion according to the specified criteria. In many ways, the second step is the most challenging. No matter how cleverly designed or diverse the starting library, without an effective screening strategy the ability to isolate useful clones is severely diminished. The best screens are (1) high throughput, to increase the likelihood that useful clones will be found;...

Adenosine 3,5 -cyclic monophosphate (cAMP) and guanosine 3,5 -cyclic monophosphate are ubiquitous nucleotides that have been described as the first and second messengers. In concert with intracellular calcium and IP3, they play a major role in the control of intracellular signaling, which orchestrates normal and pathophysiological responses. Downstream from the cyclic nucleotide synthesis by adenylyl and guanylyl cyclases, the multigenic family of cyclic nucleotide phosphodiesterases (PDEs), by specifically hydrolyzing cyclic nucleotides, controls cAMP and cGMP levels to maintain a basal...

A collection of standard and cutting-edge techniques for using Xenopus oocytes and oocytes/egg extracts to reconstitute biological and cellular processes. These readily reproducible methods take advantage of the oocyte's impressive protein abundance, its striking protein translation capacity, and its breathtaking possibilities for the assembly of infectious viral particles by single cell injection of multiple RNAs. The authors focus on the versatility of frog oocytes and egg extracts in cell biology and signal transduction, and cover all the major uses of oocytes/extracts as experimental...

A cutting-edge collection of readily reproducible techniques for the isolation, culture, and study of activation and signaling in human mast cells. These methods take advantage of the latest advances in molecular biology, technology, and information science. They include methods for the identification of mast cells, the development of mast cells in vitro, the study of mast cell signaling and gene expression, and the measurement of mast cell expression of inflammatory mediators. Additional chapters cover methods for studying mast cell interactions with other cell types (endothelial cells,...

A wide-ranging collection of readily reproducible methods for performing nuclear reprogramming by nuclear transfer in several different species, by fusion through both chemical treatment and electrically shocking cells, and by in vivo treatment of cells with cell extracts. Several methods of monitoring nuclear reprogramming are also presented, including the use of transgenic markers, activation of telomerase as an ES-specific marker, light and electron microscopic observation of structural changes in the nucleus, and verification of surface marker expression and the differentiation potential...