GO Real Estate Finance and Investment ManualAuthor: Jack Cummings
Type: eBook
Language: English
Released: 2008
Publisher: Wiley
Page Count: 714
Format: pdf
ISBN-10: 0470260408
ISBN-13: 9780470260401
Tags:Real Estate Finance and Investment Manual, tutorials, pdf, djvu, chm, epub, ebook, book, torrent, downloads, rapidshare, filesonic, hotfile, fileserve
Description:
Nucleases, enzymes that restructure or degrade nucleic acid polymers, are vital to the control of every area of metabolism. They range from "housekeeping" enzymes with broad substrate ranges to extremely specific tools. Many types of nucleases are used in lab protocols, and their commercial and clinical uses are expanding. The purpose of this book is to introduce the reader to some well characterized protein nucleases, and the methods used to determine their activity, structure, interaction with other molecules, and physiological role. Each chapter begins with a mini-review on a specific nuclease or a nuclease-related theme. Although many chapters cover several topics, they were arbitrarily divided into five sections: -Characterizing nuclease activity, which includes protocols and assays to determine general (processive, distributive) or specific mechanisms. Methods to assay nuclease products, identify cloned nucleases and determine their physiological role are also included here. -Inhibitors and activators of nucleases, summarizes assays for measuring the effects of other proteins and small molecules. Many of these inhibitors have clinical relevance. -Relating nuclease structure and function, gives an overview of methods to determine or model the 3D structure of nucleases and their complexes with substrates and inhibitors. A 3-D structure can greatly aid the rational design of nucleases and inhibitors for specific purposes. -Nucleases in the clinic summarizes assays and protocols suitable for use with tissues and for nuclease based therapeutics. -Nucleases in the lab includes protocols to use nucleases in cloning and determining the activity of other proteins. The experienced reader will immediately recognize several of the nucleases used as examples throughout this book, especially RNase A and restriction enzymes. However, new nucleases with novel specificity are constantly being discovered, performing often unexpected functions. A regulator of the unfolded protein response, identified initially as a kinase, is also a sequence specific ribonuclease (3). A human analogue of a plant ribonuclease was discovered in the search for a tumor suppressor protein (7) while angiogenin (25) was cloned as a factor stimulating blood vessel formation. RNase L is one of the mediators of Interferon activity (12). Researchers who unmask a nuclease disguised as a cytokine, mating factor, toxin, etc. should find the methods for characterizing their protein described in the first section of this book particularly useful. These chapters suggest questions to ask about the nuclease's activity or primary structure. Is the amino acid sequence novel or similar to one of the major families of nucleases (7, 18)? Is the cleavage processive or distributive, i.e., does the nuclease scan the nucleic acid polymer and cleave repeatedly before separating, as has been shown for restriction endonucleases and glycosylases (1) or, does it, in the fashion of RNase A (2), cleave and simultaneously release the polymer, generating products that are at the same time novel substrates? Does the back (synthesis) reaction affect the kinetics of the cleavage process? Is the nuclease activity essential for metabolic activity, as McClure and coworkers (5) have elegantly demonstrated for the stylar RNases? Finally, is the activity sensitive to known inhibitors or activators ? (8,9,12) The chapters at the center of this book were selected to introduce the reader to methods used to define the tertiary structure of nucleases. Of course, a complete tertiary structure determination by X-ray crystallography (13,14,17,19,20) or NMR (16) requires a good deal of time and specialized techniques . However, if the sequence has significant identity to a protein for which a structure has been determined, tools now available on the Internet allow one to model the probable 3D structure (18). The methods described aid in the design of nucleases with new properties (15,20) and improved inhibitors (13,14). Nuclease based therapies and diagnostics are coming into the clinic. DNase I therapy (20,21) has improved the lives of thousands of cystic fibrosis victims. Nucleases with demonstrated antitumor activity (24) stimulated clinical trials of other members of the RNase A family and modified forms(23, 26). Better understanding of nucleases that repair damaged DNA (1,18), mediate retroviral integration and replication (10,22), or play a role in cytokine and growth factor mechanisms (9,12,25) is important both in understanding disease progression and developing better therapeutic modalities. Antisense therapies, for example, depend on directing the activity of intracellular RNase H (11). Finally, nucleases are a major research tool in molecular biology. The exquisite specificity of restriction endonucleases (19,27,28) is routinely used in gene cloning. Exploiting the special qualities of a subclass, hapaxoterminers, can make subcloning and gene modification easier (29). There are many uses for non-specific nucleases as well. DNase I is used to locate the binding sites of proteins on DNA (30) and S1-nuclease (31) or ribonuclease (32) mapping to quantitate specific mRNAs. Degradation of nucleic acid polymers with non-specific nucleases, including DNase I, RNase A and the endonuclease from Serratia (17) are used to clarify lysates and ease protein purification. The comments and notes on the method provide guidance and insight when things go wrong (i.e., not as planned), and for how to go about correcting them. We can anticipate that the model proteins used to develop new biophysical methods and clinical therapies, which have changed little in the past 30 years, will show more variety in the future. Many new nucleases are commercially available, and the genome projects are revealing copious sequence information about nuclease families that may be more important metabolically. The examples in this book, as varied as they are, are only starting points for exploration in the wide world of nucleases. Numbers in parentheses refer to chapters in the book From the Author My suggested title for this volume was "Joy of Nucleases", and all the authors of the individual seemed to share in this feeling. RNases are no doubt a pain, if they are degrading your RNA in lab, but they can be wonderful lab and clinical tools. Inhibitors of nucleases, such as HIV-integrase, may provide new ways to control the tissue devastation caused by viruses.
